Compositions and methods for treatment of neoplastic disease

ABSTRACT

Compositions and methods for treating a tumor, neoplastic disease or infectious disease in a subject are based on superantigens in the form of polypeptides including fusion polypeptides or conjugates, homologues, and fragments, all of which induce a tumoricidal response when administered directly into tumor or an organ sheath or body cavity affected by the tumor. Nucleic acid constructs encoding the foregoing polypeptides are also used in antitumor therapy. The above agents may be administered in sustained release or controlled release vehicles at or near sites of tumors in a tumor-bearing subject.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention

[0002] The invention relates generally to immunotherapeutic compositionsand methods for treating tumors and cancer. The methods are based on theexpression of superantigen (“SAg”) alone or in combination with othermolecules in transfected host cells (tumor cells, accessory cells orlymphocytes). Other therapeutic methods are based on administering Tcells which are activated by cells engineered to express SAg and otherimmunostimulatory molecules and structures.

[0003] 2. Description of the Background Art

[0004] Therapy of the neoplastic diseases has largely involved the useof chemotherapeutic agents, radiation, and surgery. However, resultswith these measures, while beneficial in some tumors, has had onlymarginal effects in many patients and little or no effect in manyothers, while demonstrating unacceptable toxicity. Hence, there has beena quest for newer modalities to treat neoplastic diseases.

[0005] In 1980, tumoricidal effects were demonstrated in four of fivepatients with advanced breast cancer treated with autologous plasma thathad been perfused over columns in which Staphylococcal Protein A waschemically attached to a solid surface (Terman et al., New Eng. J. Med.,305:1195 (1981)). While the initial observations of tumor killingeffects with the immobilized Protein A perfusion system have beenconfirmed, some have obtained inconsistent results.

[0006] The explanation of these inconsistencies appears to be asfollows. First, commercial Protein A is an impure preparation, asevident from polyacrylamide gel electrophoresis and radioimmunoassaysthat detected Staphylococcal enterotoxins in the preparations. Second,various methods of immobilizing Protein A to solid supports have beenused, sometimes resulting in loss of biological activity of theperfusion system. Third, the plasma used for perfusion over immobilizedProtein A has often been stored and treated in different ways, alsoresulting in occasional inactivation of the system. Moreover, thesubstance(s) or factors responsible for the anti-tumor effect of thisextremely complex perfusion system have not been previously defined.

[0007] The system contained an enormous number of biologically activematerials, including the Protein A itself, Staphylococcal proteases,nucleases, exotoxins, enterotoxins and leukocidin, as well as the solidsupport and coating materials. In addition, several anaphylatoxins weregenerated in plasma after contact with immobilized Protein A. Finally,it was speculated that the biological activity of the system was due tothe removal from the plasma by the Protein A of immunosuppressive immunecomplexes that otherwise inhibit the patient's antitumor immuneresponse.

[0008] The Staphylococcal enterotoxins that contaminate the Protein Acolumns are a family of extracellular products of Staphylococcal aureusthat belong to a well recognized group of proteins that have commonphysical and chemical properties. The enterotoxins produce a number ofcharacteristic effects in humans and animals, such as emesis,hypotension, fever, chills, and shock in primates and enhancement ofgram negative endotoxic lethality in rabbits. At least some of theseeffects are due to the ability of these proteins to act as extremelypotent T cell mitogens.

[0009]Staphylococcal enterotoxins are representative of a family ofmolecules known as SAgs which are the most powerful T cell mitogensknown. They are capable of activating 5 to 30% or the total T cellpopulation compared to 0.01% for conventional antigens. Moreover, theenterotoxins elicit strong polyclonal proliferation at concentrations10³-fold lower than conventional T cell mitogens. The most potententerotoxin, Staphylococcal enterotoxin A (SEA), has been shown tostimulate DNA synthesis in human T cells at concentrations of as low as10⁻¹³ to 10⁻¹⁶M. Enterotoxin-activated T cells produce a variety ofcytokines, including IFN, various interleukins and TNF. Enterotoxinsstimulate several other cell populations involved in innate and adaptiveimmunity which also play a major role in anti-tumor immunity. Forexample, enterotoxins engage the variable region of the TCR chain onexposed face of the pleated sheet and the sides of the MHC class IImolecule.

[0010] The SAg is capable of augmenting the TH-1 cytokine response byCD4+ cells while also activating NKT and NK cells and γ/δ T cells. NKcell cytotoxicity is augmented by IFN-γ produced by SAg activated Tcells. NKT cells are known to be activated by SAgs, peptides,α-galactosylceramides and lipoarabinomannans presented on CD1 receptors.Evidence points to an invariant lectin like recognition unit on the NKTcell chain as a specific ligand for galactosylceramide determinants ontumor cells. SAgs induce tumor killing in vivo when given alone orconjugated to tumor associated antibodies. They are also effective whenemployed ex vivo to produce tumor sensitized T cells or γ/δ T cells forthe adoptive therapy of MCA 205/207 tumors. SAg transfected tumor cellshave shown a capacity to reduce metastatic disease in a murine mammarycarcinoma model.

[0011] In addition to these common biological activities, theStaphylococcal enterotoxins share common physicochemical properties.They are heat stable, trypsin resistant, and soluble in water and saltsolutions. Furthermore, the Staphylococcal enterotoxins have similarsedimentation coefficients, diffusion constants, partial specificvolumes, isoelectric points, and extinction coefficients. TheStaphylococcal enterotoxins have been divided into five serologicaltypes designated SEA, Staphylococcal enterotoxin B (SEB), Staphylococcalenterotoxin C (SEC), Staphylococcal enterotoxin D (SED), andStaphylococcal enterotoxin E (SEE), which exhibit striking structuralsimilarities. The enterotoxins are composed of a single polypeptidechain of about 30 kilodaltons (kD). All staphylococcal enterotoxins havea characteristic disulfide loop near the middle of the molecule. SEA isa flat monomer consisting or 233 amino acid residues divided into twodomains. Domain I comprises residues 31-116 and domain II of residues117-233 together with the amino tail 1-30. In addition, the biologicallyactive regions of the proteins are conserved and show a high degree ofhomology. One region of striking amino acid sequence homology betweenSEA, SEB, SEC, SED, and SEE is located immediately downstream (towardthe carboxy terminus) from the cysteine located at residue 10⁶ in SEA.This region is thought to be responsible for T cell activation. A secondhomologous region that begins at residue 147 and extends downstream ishighly conserved. This region is believed to mediate emetic activity.The region related to emetic activity can be omitted from enterotoxinsused as therapeutics.

[0012] A sequence analysis of the Staphylococcal enterotoxins with othertoxins has revealed SEA, SEB, SEC, SED, Staphylococcal toxicshock-associated toxin (TSST-1 also known as SEF), and the Streptococcalexotoxins share considerable nucleic acid and amino acid sequencehomology. The enterotoxins belong to a common generic group of proteinsthought to be evolutionarily related.

[0013] Enterotoxins bind to MHC Class II molecules and the T cellreceptor (“TCR”) in a manner quite distinct from conventional antigens.Enterotoxins engage the variable region of the TCR β chain on an exposedface of the β pleated sheet and the sides of the MHC Class II molecule,rather than engaging the groove of the Class II molecule likeconventional antigens. In contrast to SEB and the SEC, which have onlythe capacity to bind to the MHC class II α chain, SEA, as well as SEEand SED, also interacts with the MHC class II β chain in a zincdependent manner T cell recognition is based on the presence of the βchain and is therefore independent of other TCR components and diversityelements. Single amino acid positions and regions important for SAg-TCRinteractions have been defined. These residues are located in thevicinity of the shallow cavity formed between the two domains. Thealanine substitution of amino acid residue Asn23 in SEB has demonstratedthe importance of this residue in SEB/TCR interaction. This particularresidue is conserved among all of the Staphylococcal enterotoxins andmay constitute a common anchor position for enterotoxin interaction withTCR Vβ chains. Amino acid residues in positions 60-64 have also beenshown to contribute to the TCR interaction as do the cysteine residuesforming the intermolecular disulfide bridge of SEA. For SEC2 and SEC3,the key points of interaction in the Vβ chain are located in the CDR1,CDR2 and HRV4 TCR Vβ-3 chain. Hence, multiple and highly variable partsof the Vβ chain contribute to the formation of the enterotoxin bindingsite on the TCR. Thus far, a single and linear consensus motif in theTCR Vβ displaying a high affinity interaction with particularenterotoxins has not been identified. A significant contribution of theTCRα chain in enterotoxin-TCR recognition is acknowledged as well as MHCclass II isotypes. This distinctive binding mechanism of enterotoxinswhich bypasses the highly variable parts of the MHC class II and TCRmolecules allows them to activate a high frequency or T cells withmassive lymphoproliferation, cytokine induction and cytotoxic T cellgeneration. These properties are shared by other proteins made byinfectious agents. Together, these proteins form a well recognized groupknown as SAgs.

[0014] There are two general classes of SAgs. The first includes minorlymphocyte stimulating (MLS) antigens. The second class of SAgs includesmycoplasmal, viral, and bacterial proteins such as the Staphylococcalenterotoxins and streptococcal exotoxins. All SAgs have the followingproperties. T cell activation does not require antigen processing. Thereis no MHC restriction of responding T cells. SAgs bind to and evokeresponses from all T cells expressing V receptors, without requiringother TCR or diversity elements. CD4-CD8-α/β+T cells and γ/δT cells arealso capable of responding to SAgs by proliferating, producing TH1cytokines and exhibiting cytotoxic activity. The SAgs induce abiochemically distinct T cell activation pathway. Thus, SAgs interactwith and activate a much larger proportion of T cells than conventionalantigens, causing massive lymphoproliferation, cytotoxic T cellgeneration, and cytokine secretion. A given SAg can activate up to 30%of resting T cells compared to 0.01% for conventional antigens. Ashighly representative members of this family of SAgs, the enterotoxinsshare these characteristics.

[0015] The present invention features the use of SAgs in associationwith molecules to produce tumor killing effects. The SAgs are useful inpeptide form and may combine with another peptide or nucleic acid toform a conjugate. The effect of the combined molecules is synergistic.These conjugates are useful when administered as a preventative ortherapeutic antitumor vaccine in tumor bearing patients. Alternatively,they may be used ex vivo to load an antigen presenting cell as a meansof immunizing a T. γ/δ, NK or NKT cell population for use in adoptivetherapy of cancer. Examples of such conjugates are complexes between:SAg and glycosylceramide; SAg and apolipoproteins (Lp(a)), SAg andoxyLDL, SAg and verotoxins, SAg and GPI-ceramide (with phytosphingosinebackbone), SAg and lipopolysaccharide (LPS), SAg and peptidoglycan, SAgand mannan protcoglycan, SAg and muramic acid, SAg andphytosphingolipid, SAg and tumor peptides. Also intended are SAg and Galconjugates and glycosylated SAgs.

[0016] The present invention features the use of SAg in association orconjugated to oxidized low density lipoproteins (oxyLDL) andapolipoproteins (e.g., lipoprotein (a) (Lp(a)). OxyLDL and itsbyproducts bind to receptors on sinusoidal endothelial cells in thetumor microcirculation where they induce apoptosis, increase levels oftissue factor and activated thrombin, upregulate adhesion molecules andproduce a prothrombotic state. Lp(a) is densely deposited in tumormicrocirculation and as a competitive inhibitor of plasminogen isprothrombotic. Hence, both apolipoproteins and oxyLDL not only home toreceptors on the tumor microcirculation but they also induce endothelialcell or macrophage apoptosis as well as a prothrombotic state. Theselocal effects are amplified by the presence of the conjugatedsuperantigen which induce a localized T cell immune and inflammatoryresponse collectively resulting in a potent anti-tumor response.

[0017] The present invention also features the use of the SAg inassociation or conjugated to verotoxins. The latter molecules have thecapacity to bind to galactosylceramide receptors on tumor cells andinduce apoptosis. Hence, the tumor targeting and apoptosis inducingfunctions of the verotoxin are coupled with the T cell immune andinflammatory response induced by the SAgs to produce a potent and welllocalized anti-tumor response.

[0018] The present invention features the use of SAgs in association orconjugated to mono or digalactosylceramides. The latter have beenisolated from human kidney, Fabry's disease kidney, marine spongeAegelus mauritanius and is expressed in certain bacteria such asSphingomonas paucimobilis. They have been shown to activate NKT cellsand to induce anti-tumor effects in vivo against several types oftumors. The activation of NKT cells in the presence of the mono anddigalactosylceramides appears to be IL-12 dependent. The biologicalactivity of the -galactosylceramides is observed in both mono anddigalactosylceramide forms and is dependent upon the presence of ananomeric configuration on the terminal galactose. The lengths of thesphingosine base and fatty acyl chains of 23 and 15 respectively alsoappear to be optimal for production of the anti-tumor effects. SAg isalso used in association with phytosphingosine which is expressed inSaccharomyces cerevisiae membranes and vesicles.

[0019] SAgs are known to be the most powerful T cell mitogens known andhave been shown to produce anti-tumor effects in several animal models.The -galactosylceramides are known to be potent inducers of NKT cellactivation which have been shown to produce an anti-tumor effect in anIL-12 dependent manner. In the present invention SAgs are combined with-galactosylceramides biochemically as conjugates and genetically withina cell which expresses the newly synthesized protein-boundgalactosylceramide on the cell surface. The newly synthesized conjugatesin native form or expressed in or on the cell produce a synergisticanti-tumor effect due to the activation of T cells and NKT cellpopulations.

[0020] Furthermore, in the present invention the SAg-galactosylceramidesare expressed in tumor cells, dendritic cells (“DC”) or a hybrid cellmade by fusing a tumor cell and a DC. The use of DCs or DC/tumor cellhybrids (DC/tc) to present the SAg-galactosylceramides fusion constructsor conjugates provides the optimal costimulation for activation of atumor specific T cell population. The use of a tumor cell or a DC/tcprovides in addition to costimulation, expression of the tumor antigen,itself to activate anti-tumor T and NKT cell clones which are tumorspecific. Hence, an optimal cell is a DC/tc which expresses SAg andSAg-anomeric galactosylceramides.

[0021] The SAg-galactosylceramide conjugates are useful in the presentinvention. However, there are distinct differences and advantages toproducing and expressing the SAg-αgalactosylceramide conjugates within acell. First, final products are quite different. One involves theenterotoxin-α-galactosylceramide in free form whereas the other involvescell associated enterotoxin-αgalactosylceramide which includesenterotoxin nucleic acids and peptides. In the cell both enterotoxinsand α-galactosylceramides are associated with numerous intracellular andmembrane structures such as MHC, costimulatory and adhesion molecules,heat shock proteins, membrane glycolipids and glycosphingolipids whichmay improve immunogenicity and antigen presentation. They may also betransported in various vesicles and exosomes which may provideadditional immunogenicity. With the addition of appropriate signalssequences and association with molecules involved in the antigenpresenting pathways such as the invariant chain, TAP and LAMP molecules,the conjugates may be routed in the cell to the MHC class I, class II orCD1 receptor. Therefore, enterotoxin and α-galactosylceramides producedwithin a cell are presented to the host's immune system in an entirelydifferent form compared to the purified enterotoxin polypeptide.

[0022] Unlike free enterotoxin polypeptide or -galactosylceramide, SAgtransfected tumor cells, DCs or DC/tc present enterotoxins to the T cellsystem in association and or conjugated to tumor associated antigensincluding mutated normal structures or fusion structures, costimulatoryand adhesion molecules. Indeed, the coadministration of SAg with tumorantigen would be expected to produce a heightened response to the tumorantigens while preventing the clonal deletion which occurs with SAgalone. Liu et al., Proc. Natl. Acad. Sci., 88: 8705-8709, (1991);McCormack et al., Proc. Natl Acad. Sci., 91: 2086-2090, (1994); Coppolaet al., Int. Immunol., 9: 1393-403, (1997). Hence, the coadministrationof SAg-galactosylceramide and tumor associated antigens would induce apredictably heightened tumor specific response by the host. Thisprediction was borne out by the Applicant's work showing that SAgtransfection of tumor cells abolished the tumorigenicity of 4T 1 mammarycarcinoma cells, significantly reduced the number of establishedmetastases and prolonged survival compared to untreated controls.(Pulaski, Terman, et al., American Association of Cancer Research, April1999; Cancer Research in press 2000).

[0023] SAg transfected tumor cells in vivo are effective in anadditional manner which does not apply to SAg polypeptide. Ingestion ofapoptotic cells by DCs augments the immunogenicity of tumor cells.Fields et al., Proc. Natl. Acad. Sci., 95: 9882-9887, (1998); Albert etal., Nature, 392: 86-89, (1998). DCs are acknowledged as the premieraccessory cell for antigen presentation. They have been shown to ingestapoptotic cells and nucleic acids and process them for presentation tohost T cells in the context of costimulation, adhesion and MHCmolecules. Akbari et al., J. Exp. Med., 189: 169-177, (1999). Therefore,following apoptosis of SAg transfected tumor cells and ingestion by DCs,SAg-encoding nucleic acid as well as tumor associated nucleic acids inthe transfected cells would produce additional anti-tumor responses.Purified polypeptide enterotoxins do not share with the SAgtransfectants this property of enhanced immunogenicity followingingestion and processing by DCs.

[0024] There are enormous structural and functional differences betweenthe polypeptide enterotoxin and SAg-transfected tumor cells. Thestarting materials are different, i.e., peptides vs nucleic acids andthe product is different, i.e., polypeptide vs enterotoxin transfectedcell in which the SAg is may exist in nucleic acid and peptide formassociated with a vast number of intracellular and membrane structures.Some of these structures may actually improve the T cell activatingfunction of SAgs such as deoxyribonucleic acids, ribonucleic acids,tumor associated antigens, heat shock proteins, costimulatory moleculesand adhesion molecules and endosomes. Cellular SAg peptides ornucleotides exist in association with tumor associated antigens,costimulants, adhesion molecules, heat shock proteins and MHC molecules,GPI-ceramides or SAg receptors (digalactosylceramides) which improve theimmunogenicity of the tumor antigens. Therefore, these structural andfunctional differences between the polypeptide SAg and the enterotoxintransfected tumor cells clearly show that SAg transfected tumor cellshave a far greater potential than the polypeptide to induce a tumorspecific response.

[0025] Moreover, SAg transfected tumor cells possess an additionalunique property not shared by the polypeptide SAg. SAg-transfected tumorcells display the metastatic phenotype of the tumor cells which enablesthem to colonize and traffic to metastatic sites in vivo. Once localizedto micrometastatic sites the transfectants expressing SAg induce apotent tumor specific T cell response. In contrast, the purifiedpolypeptide SAg unassociated with a tumor cell would have no capacitywhatsoever to colonize metastatic sites.

[0026] The present invention also provides SAg-encoding nucleic acid,preferably DNA, fused with (or cotransfected with) a nucleic acidencoding another molecule. The transfected cells include tumor cells,accessory cells e.g., DCs, tumor cell/accessory cell (e.g., DC) hybrids.The expression of molecules in addition to enterotoxins by these cellsserves the following function:;

[0027] 1) enhance the immunogenicity of the SAg transfected cell byproviding nucleic acids encoding an additional potent immunogen.Examples would include tumor associated antigens or mutated normalantigen or fusion peptides in tumor cells, an immunogenic bacterialproduct such as Staphylococcal adhesin protein A, LPS, β-glucans, andpeptidoglycans, costimulatory and adhesion molecules, heat shockprotein, growth factor receptors such as Her/neu and tumor markers suchas PSA.

[0028] 2) assist in tumor killing activity by the SAg transfected cellwhen localized to tumor sites by providing nucleic lacids encoding thefollowing: angiogenesis antagonists, chemoattractants such as C5a,chemokines such as RANTES, hyaluronidase and coagulase and CD44isoforms.

[0029] 3) Increase the binding of immunogenic substances to the surfaceof the SAg transfected cell by providing nucleic acids encoding thefollowing: CD1 receptors, CD14 receptors, SAg receptors

[0030] 4) increase the production of SAg in the SAg transfected cell byproviding nucleic acids encoding the following: cell cycle proteins,amplified oncogenes, and signal transduction molecules.

[0031] 5) assist in trafficking of SAg to class I or class II pathway inthe SAg transfected cell by providing nucleic acid encoding thefollowing: the invariant chain, the LAMP1 proteins and TAP proteins.

[0032] 6) induction of a local tumoricidal response by intratumoralinjection of nucleic acids encoding the following: oxyLDL receptor andSAg receptor, chemoattractants, chemokines.

[0033] The present invention also provides for augmented tumoricidalresponses by immunocytes particularly T, γ/δ T, NK, and NKT cells.Inhibitory receptors or their tyrosine-based inhibitory motifs on T, γ/δT, NK, and NKT cells with specificity for lipid-based tumor associatedantigens (LBTAAs) are deleted or functionally deactivated (antisense orgene knockout) which permits unopposed intracellular signaling by theactivation receptors and enhanced responsiveness to LBTAAs and theirrespective tumors of origin. Inhibition of inhibitory receptorphosphorylases (SHP or SHIP) and/or ITIM binding sites on activationreceptors (ITAM) is also contemplated as a means of augmenting the hostresponse to LBTAAs.

SUMMARY OF THE INVENTION

[0034] The present invention comprises a method for treating cancer in ahost comprising providing conjugates, fusion proteins or naked nucleicacids of superantigen and additional molecule(s) which produce antumoricidal response. The additional molecule serves the followingfunctions: 1) to target a receptor (digalactosylceramide) expressed ontumor cells in vivo and induce tumor cell apoptosis e.g., SAg-verotoxinconjugates. 2) to target receptors expressed on tumor sinusoidalendothelium, induce apoptosis and a prothrombotic state e.g. SAg-oxyLDLconjugates and SAg-Lp(a) conjugates 3) to activate a dormant populationof tumoricidal NKT cells e.g. SAg-digalactosylceramides,SAg-GPI-digalactosylceramide (phytosphingosine) complexes. 4) targetreceptors for integrins expressed on tumor microvasculature e.g.,SAg-RED conjugates. 5) naked DNA administered intratumorally inducestumor cell expression in vivo of receptors for ligands which produceapoptosis and inflammation e.g., naked DNA SAg-oxyLDL receptor,SAg-LOX-1 receptor, SAg-SREC receptor.

[0035] Sickled erythrocytes are useful in the present invention sincethey have natural ligands for integrins expressed on tumorneovasculature which facilitates their targeting to the tumorendothelium. Sickled erythrocyte membranes acquire oxyLDL usingfusigenic techniques with oxyLDL containing liposomes and apoproteinsvia gene transfection in the nucleated pre-reticulocyte phase. TheoxyLDL and apoproteins expressed by the sickled cells facilitatestargeting to oxyLDL, LOX-1 and SREC receptors present on the tumormicrovasculature. These erythrocytes are also useful for carryingnucleic acids for transfection of the tumor endothelial cells in vivo.Vesicles derived from sickled erythrocytes are more rigid, prothromboticand target the tumor microvasculature more effectively than the parentcell. They also carry oxyLDL to receptors on tumor endothelium.Likewise, vesicles, exosomes or SAg-GPI-digalactosylceramides shed fromSAg transfected tumor cells are capable of inducing potent tumoricidalresponses and are useful in the present invention.

[0036] In addition, bacterial and yeast expression and phage displaysystems are useful for the presentation of SAg in association with otheranti-tumor molecules. The yeast sec mutant or yeast display is used toproduce a SAg-ceramide conjugate exhibiting a phytosphingosine in thesphingosine portion of the ceramide. This structure activates both Tcell, γ/δ T, NK cell and NKT cells. Sphingomonas paucimobilis whichnaturally expresses a α-galactosylceramide is transfected with SAgnucleic acids which results in the shedding of SAg-α-galactosylceramidecomplexes which are use to produce a population of tumoricidal T cells,γ/δT, NK cells and NKT cells. SAg phage displays with tumor localizingmolecules e.g. RED sequences are used to target SAgs to tumormicrovasculature. SAg phage displays with similar tumor localizingmolecules comprising tumor or tumor endothelial cell apoptosis inducingagents e.g., thrombospondin or oxyLDL are use to increase thetumoricidal response.

[0037] The present invention comprises a method for treating cancer in ahost comprising providing cells transfected with a gene that expressand/or secretes a SAg or T cells activated by the transfected cells tothe host. The cells are transfected in vivo or in vitro. SAgs mayactivate T cells, γ/δ T or NKT or NK cells in the host. These sametransfectants may be used to stimulate a population of T cells, γ/δ Tcells or NKT cells ex vivo which are provided to the host as tumorspecific effector cells in adoptive immunotherapy. The transfected cellsmay be, for example, tumor cells accessory cells, DCs muscle cells,immunocytes, fibroblasts. When transfected in vitro the cells can bexenogeneic to the host, from the same species as the host or host cells.

[0038] For in vivo immunization, tumor cells are transfected withnucleic acids encoding SAgs together with a carbohydrate modifyingenzyme such as α-galactosyl transferase to produce the α-Gal epitope,Staphylococcal hyaluronidase, Streptococcal capsular polysaccharide,Staphylococcal erythrogenic toxin, Staphylococcal Protein A,Staphylococcal βhemolysin, Staphylococcal coagulase, costimulants suchas B7-1 and B7.2, chemoattractants and chemokines. SAgs are alsocotransfected into tumor cells with gene clusters encoding thebiosynthesis of highly immunogenic microbial Lipid A, membrane orcapsular polysaccharides, lipoproteins and peptidoglycans. Nucleic acidsare useful when transfected alone. However combinations are preferred.The cotransfection into tumor cells of the SAg-encoding nucleic acidtogether with the nucleic acids encoding αGal or GalCer biosynthesis isparticularly useful. The cotransfection into tumor cells of the nucleicacid encoding SAg with nucleic acids encoding Staphylococcalerythrogenic toxins and hyaluronidase allows the transfected tumor cellsto simulate the in vivo inflammatory activity of a Staphylococcus orleukocyte or macrophage by secreting enzymes and toxins which induce asterile cellulitis in tumor sites.

[0039] Further provided are tumor cells transfected with nucleic acidencoding structures such as the erb/Neu gene which upon administrationto the host promotes tumor cell trafficking and colonization ofmicrometastatic sites. Amplified oncogenes linked to SAg nucleic acidsprovide the locus and energy for expression or overexpression of bothgene products. Thus, provided herein are tumor cells transfected withSAg-encoding nucleic acid together with nucleic acid encoding otheroncogenes, amplified oncogenes and transcription factors, angiogenicfactors such as angiostatin, angiogenesis receptors such as VEGF, tumorgrowth factors, tumor suppressors, cell cycle proteins and key proteinsengaged in the antigen routing and processing pathway. In one example,the microbial SAg and erb/Neu nucleic acids are cotransfected into tumorcells. These nucleic acids may also linked to an inducible gene such asthat encoding metallothionein or corticosteroid receptors. In this way,the cells are activated by exogenous delivery of corticosteroids or aheavy metal only after a suitable period of time has lapsed to allowthem to localize in metastatic sites in vivo

[0040] Tumor cell transfectants are also useful ex vivo to immunize a Tcell, γ/δ T cell or NKT cell population producing tumor specificeffector cell population for adoptive immunotherapy of cancer. Theseimmunizing tumor cells are transfected with nucleic acids encoding SAgsand the SAg receptor. The latter transfectants are capable of bindingexogenous SAg for presentation to a T cell population. In addition,tumor cells are transfected with nucleic acids encoding CD1 receptorswhich are capable of binding exogenous glycosylceramides andlipoarabinans free or bound to SAgs for presentations to T, γ/δ or NKTcells. Similarly, tumor cells are transfected with nucleic acidsencoding the CD14 receptor which bind exogenous peptidoglycans andLPS's, free or bound to SAgs for presentation to T cells.

[0041] Likewise, the nucleic acids encoding the mannose receptor aretransfected into tumor cells which are capable of binding a broad rangeof glycosylated SAgs for presentation to T cells. The present inventionprovides detailed methods for preparation of the SAg-glycosylceramide,SAg-LPS, SAg-peptidoglycan complexes as well as glycosylated SAgs whichare loaded onto their respective receptors expressed on tumor cells,accessory cells and, in some instances, immunocytes. For ex vivo use,any prokaryotic or eukaryotic cell may be used which is transfectablewith nucleic acid encoding SAgs to provide surface expression of the SAgor constructs expressed on tumor, accessory cell or immunocytetransfectants. When the transfected cells are not host tumor cells, thecells additionally express a tumor associated antigen expected to bepresent on the host's cancer cells.

[0042] Also provided is a tumor specific T cell, γ/δ, NK or NKT cell(collectively immunocyte) population which is activated by SAgs, SAgsconjugates given above or the tumor cell transfectants given above toproduce a population of tumor specific effector cells useful in adoptiveimmunotherapy. A particularly effective method of producing ahyperresponsive immunocyte population is to delete (e.g. gene knockout)or inactivate (e.g. antisense) receptors on immunocytes or theirrespective immune receptor tyrosine-based inhibitory motifs (ITIMs)which inhibit cellular activation by receptors specific for lipid-basedtumor associated (Lip-TAAs) and/or superantigens. After exposure toLip-TAAs and/or superantigens, the immunocyte activation receptorresponse is unopposed by an inhibitory signal in which case immunocytesreadily differentiate into tumor specific effector cells which arehighly reactive even to weak Lip-TAAs.

[0043] After ex vivo stimulation, the T cells, γ/δ T cells or NKT cellsused for adoptive immunotherapy should preferentially express CD44 whichindicates that they are capable of trafficking and homing to tumorsites. Additionally, the T cell population used for ex vivo immunizationis engineered to overexpress the TCR variable Vβ and invariant Vα sitesspecific for SAg and glycosylceramide binding respectively and toproduce IFN by exogenous delivery of corticosteroids or a heavy metal. Aparticularly useful population of therapeutic tumor specific effector Tcells or NKT cells which demonstrates overexpressed CD44 together withVβ variable and Vα invariant regions and high IFN production. Alsoprovided are methods for reactivating anergic T cells in cancer patientsby transfecting nucleic acids encoding the SAg receptors to produce a Tcell population which may now be stimulated with exogenous SAgs.

[0044] Compositions which mimic SAgs are used in place of native SAgsfor in vivo administration in order to circumvent the problem ofnaturally occurring SAg-specific antibodies. The SAg mimics are largelycomprised of nucleotides or oligonucleotide-peptide chimeric constructswhich are specific for tumor cells expressing SAg receptors (via thenucleotide) while retaining their SAg specificity for the TCR (via thepeptide). The class II binding site of the SAg may optionally beeliminated or mutated to minimize SAg peptide binding to MHC class IIreceptors in vivo. The molecule may be composed entirely of nucleotidesfor which there are no naturally-occurring antibodies. In addition,carriers are provided for in vivo transfection of tumors by nucleicacids encoding SAgs or other nucleic acid constructs given in Table I.Phage displayed tumor neovasculature ligands may also carry nucleicacids encoding SAgs or other constructs.

[0045] The constructs and method are used to treat any solid tumor suchas carcinoma, melanoma and sarcoma or cancer of hemopoietic origin, suchas lymphomas and leukemias which may or may not form solid tumors.

[0046] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art of this invention. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting. Other features and advantages of the invention will beapparent from the following detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0047]FIG. 1. Schematic diagram of the cloning of the SEB gene into thepHβ Apr1-neo vector. The coding region of the SEB gene was amplifiedwith PCR primers. The upstream primer (SEB1) has a SalI site at its 5′end and the downstream primer (SEB2), a BamHI site. Both the pHβApr1-neo vector and the amplified SEB insert were digested with SalI andBamHI, ligated and transformed into XL1-Blue competent cells. The finalconstruct was verified by restriction enzyme and sequence analyses.

[0048]FIG. 2. Cloning of the SEB gene into the pHβ-Apr1 neo vector.Clones 1-5 contained the SEB insert (coding region 801 bp) and the pHβ-Apr1 neo vector (10 kb). All DNA was digested with SalI and BamHI andelectrophoresed on a 1% agarose gel in 1× TAE buffer.

[0049]FIG. 3. Protection of mice from tumor growth by DNA immunizationwith nuclcic acid encoding a fusion of human papilloma virus HPV16oncoprotein (E7) with SEB. C57BL/6 mice were immunized i.d. by particlebombardment (gene gun) with control vector, E7 alone, SEB-E7 and E7-SEBfusion genes. Mice were challenged with syngeneic TC-1 tumor cellstransfected with E7 or another HPV oncoprotein, E6. Mice receiving theSEB-E7 fusion gene showed complete protection against challenge. Micereceiving E7-SEB (fusion protein in reverse order), E7 only, SEB andvector all developed tumors.

[0050]FIG. 4. Protection of rabbits from growth of papilloma tumorcaused by cottontail rabbit papillomavirus (CRPV). Inbred EIII/JCrabbits were immunized with DNA by the route of FIG. 3. Groups weregiven CRPV E1 or E6, DNA, SEB DNA, and fusions of SEB with E1 or E6.Rabbits were challenged with CRPV and tumor development was monitored.The SEB-E1 fusion DNA was the most effective in inhibiting the growth ofthe outgrowth of CRPV-induced papillomas.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0051] The present invention provides methods and materials for treatingcancer related to the polypeptide or nucleic acid conjugates or fusionscomprising SAg with other molecules that synergize or cooperate with SAgin the induction of an anti-tumor response. The present invention alsoprovides materials and methods for treating cancer related totransfection of cells with nucleic acid that encode a SAg and/or anotherpolypeptide. The cells can be transfected in vivo or in vitro. Theexpression of the SAg polypeptide activates host immunocytes, such asvarious T cell subsets, including γ/δ T cells, as well as NK and NKTcells.

[0052] As used herein, T cells are defined as any class of lymphocytesthat undergo maturation and differentiation in the thymus. They mayexpress various chains of the T cell receptor (TCR). They include, butare not limited to NK cells, NKT cells and TCR α/β+ T cells, TCR γ/δ+ Tcells and may be known as cytotoxic T cells, T helper cells, suppressoror regulatory T cells, or they may be defined by the expression or typeof T cells markers (e.g., CD3, CD4, CD8) or TCR chains that theyexpress.

[0053] Various useful therapeutic constructs involving superantigencompositions, and some of the preferred conditions of use are listed inTable I, below. TABLE I Therapeutic Constructs and Preferred Conditionsof Use I. CELLS: Tumor Cells, DCs or DC/Tumor Cell Hybrids (DC/tc) USE:In Vivo and Ex Vivo PURPOSE: A. In vivo Preventative or TherapeuticVaccine (Established Tumor) Accomplish by transfecting orco-transfecting with nucleic acid encoding superantigen plus one or moreof the following: 1. Superantigens (SAgs) 2. Enzyme that modifiescarbohydrate to induce Gal or GalCer epitope expression 3. Functionalhyaluronidase from microbial or human sources 4. Staphylococcal orstreptococcal erythrogenic toxin 5. Staphylococcal protein a or a domainthereof 6. Staphylococcal hemolysin and functional microbial toxins 7.Functional microbial or human coagulase 8. Costimulatory protein 9.Chemoattractants 10. Chemokines 11. Nucleic acids encoding biosynthesisof lipopolysaccharides 12. Nucleic acids encoding biosynthesis ofglycosylceramides 13. Nucleic acids encoding biosynthesis of microbialmembrane or capsular lipoproteins and polysaccharides 14. Oncogenes,amplified oncogenes and transcription factors 15. Angiogenic factors andreceptors 16. Tumor growth factor receptors 17. Tumor suppressorreceptors 18. Cell cycle proteins 19. Heat-shock proteins, ATPases and Gproteins 20. Proteins engaged in antigen processing, sorting andintracellular trafficking 21. Inducible nitric oxide synthase (iNOS) 22.apolipoproteins (e.g., Lp(a)) transfected into tumor cells & sicklederythrocytes used for targeting tumor microvasculature 23. LDL andoxyLDL receptors (e.g., SCEP receptor) transfected into tumor cells andsickled erythrocytes & used for targeting to tumor microvasculature B.Ex Vivo Immunization of T, y/δT or NKT cells to Produce Tumor SpecificEffector Cells (for Adoptive Immunotherapy)* Accomplish by (i)transfecting or co-transfecting tumor or accessory cells with nucleicacid encoding the following, or (ii) providing immobilized molecules orreceptors that present the following: 1. Superantigen 2. SAg receptorand transcription factor with bound SAg 3. CD1 receptor binding and/orexpressing SAg-glycosyl ceramide complex 4. CD14 receptor binding orexpressing SAg-lipopolysaccharide or SAg-peptidoglycan complex 5.Mannose receptor binding glycosylated SAg 6. Glycophorin receptor 7.SAg-tumor peptide(s) complex on MHC or CD1-bearing APC in soluble orimmobilized form C. Therapeutic Molecules or Complex Applied toTransfected or Untransfected Tumor cells or Accessory Cells; or MHCclass I, class II, CD1, SAg receptor or CD14 receptor: 1. SAg (whereincell may express Gal) 2. Glycosylated SAg 3. SAg complex with a.glycosyl ceramide b. lipopolysaccharide c. peptidoglycan d. mannanproteoglycan e. muramic acid f. tumor peptide g. glycosylceramides withterminal Gal(□1-4)Gal e.g. globotriosylceramide and galabiosylceramideh. Conjugates of SAg-(Gb2 or Gb3 or Gb4) i. Conjugates of SAg-(Gb2 orGb3 or Gb4)-CD1 j. GPI anchored conjugates: SAg-GPI-(Gb2 or Gb3 or Gb4)l. GPI anchored conjugates: SAg-GPI-(Gb2 or Gb3 or Gb4)-CD1 m.Conjugates of SAg polypeptide or nucleic acid with Verotoxin n.Conjugates of SAg polypeptide or nucleic acid with Verotoxin A or Bsubunit o. Conjugates of SAg polypeptide or nucleic acid with IFNαreceptor peptides homologous to verotoxin p. Conjugates of SAgpolypeptide or nucleic acid with CD19 peptides homologous to verotoxinq. Conjugates of SAg polypeptide or nucleic acid with Arg-Gly-Asp orAsn-Gly-Arg r. Conjugates of SAg polypeptide or nucleic acid with LDL,VLDL, HDL s. Conjugates of SAg polypeptide or nucleic acid withApolipoproteins (e.g., Lp(a), apoB-100, apoB-48, apoE) t. Conjugates ofSAg polypeptide or nucleic acid with oxyLDL, oxyLDL mimics, (e.g.,7β-hydroperoxycholesterol, 7γ-hydroxycholesterol, 7-ketocholesterol,5α-6α- epoxycholesterol, 7β-hydroperoxy-choles-5-en-3β-ol,4-hydroxynonenal (4-HNE), 9- HODE, 13-HODE and cholesterol-9-HODE) u.Conjugates of SAg polypeptide or nucleic acid with oxyLDL byproducts(e.g. lysolecithin, lysophosphatidylcholine, malondialdehyde,4-hydroxynonenal) v. LDL and oxyLDL receptors (e.g., LDL oxyLDL,acetyl-LDL, LDL, LRP, CD36, SREC, LOX-1, macrophage scavenger receptors)as polypeptide or nucleic acid alone or with SAg polypeptide or nucleicacid intratumorally w. phytosphingosine, -GPI-phytosphingosine x tumorassociated lipid antigens, glycolipid, proteolipid, glycosphingolipid,sphingolipid with inositol phosphate -containing head groups,phytoglycolipids, mycoglycolipids. GPI-sphingosines or lipids y.sphingolipids with inositol phosphate-containing head groups having thegeneral structure: ceramide-P-myoinositol-X where X is a polarsubstituent such as ceramide-p-inositol-mannose,inositol-1-P-(6)mannose(a1,2-inositol-1P-(1) ceramide,(inositol-P)₂-ceramide, inositol-P-inositol-P-ceramide,inositol-P-inositol-P- ceramide. z. tumor associated glycan antigens -peptidoglycans or glycan phosphotidylinositol (GPI) structures. aachemokine receptors polypeptide or nucleic acid II. CELLS: SpecializedTumor Specific Effector Cells (T, y/δT, NK or NKT cells) USE: AdoptiveImmunotherapy In vivo PURPOSE: A. CD44 Expression on T cells or NKT --accomplished by: (i) SAg stimulation; and/or (ii) transfection withnucleic acid encoding CD44 and/or (iii) transfection with nucleic acidencoding glycosyltransferase B. Chimeric TCR with Invariant a chain sitefor binding GalCer and Vβ chain site for binding SAg C. Dual TCR Vβchains with sites for SAg binding D. T cells or NKT cells withoverexpressed Vβ region specific for a given SAg E. T cells or NKT cellswith lowered signal transduction threshold III. MOLECULES: SAg mimicsUSE: In Vivo Administration A. SAg receptor-binding oligonucleotides B.SAg oligonucleotide-peptide conjugate; oligonucleotide is ‘specific” forSAg receptor on tumor cells Peptide has deleted class II binding siteand intact TCR binding site C. Phage displayed integrin ligand on tumorneovasculature - carrier for SAg-encoding nucleic acid. IV. CARRIERS fornucleic acid encoding SAg USE Transfection of Tumors In vivo A. Sicklederythrocytes that target tumor neovasculature B. Phage displayed tumorneovascular integrin and SAg receptor carrying SAg nucleic acids V.CARRIERS constructed to co-express SAg conjugates or complexes withglycosylceramide, αGal, lipopolysaccharides, peptidoglycans USETransfection of Tumor Cells and/or DCs and/or DC/tc's - in vivo or exvivo. A. Liposomes B. Proteosomes

[0054] A single transfecting nucleic acid molecule, or a separatenucleic acid molecule, can also encode another polypeptide such as anadhesion molecule, glycosyltransferase, glycosidase, CD44, cytokine,tumor associated antigen, costimulatory molecule, and the like. Inaddition, cells transfected in vitro or ex vivo with any of thesenucleic acids as well as T cells activated by these transfected cellsare administered directly to a cancer-bearing host. Cells transfected invitro or ex vivo as well as cells activated ex vivo may additionallyexpress a tumor associated antigen expected to be present on host cancercells.

[0055] Further, cells transfected with nucleic acid that encodes a SAgpolypeptide is also be used as a vaccine to immunize a host against acancer previously present in the host or a cancer that is likely todevelop in the host. For example, a host can be vaccinated against aparticular cancer by administering tumor cells transfected with nucleicacid encoding a SAg. Alternatively, a SAg transfected cell is used toactivate a host T cell population in vitro. This activated T cellpopulation is then administered to a host as a cancer treatment(immunotherapeutic agent). Once activated ex vivo or in vivo, these Tcells are expanded with cytokine treatment such as IL-2 treatment.

[0056] Cells to be “transfected” include accessory cells, immunocytes,fibroblasts, or tumor cells. Accessory cells may include, withoutlimitation, endothelial cells, DCs, monocytes, macrophages as well as Band T lymphocytes which can play an “accessory” as well asdirect-effector role in an immune response. When transfected in vitro,the cells can be xenogeneic, allogeneic to the host to provide, amongother things, additional immunogenicity. Preferably, the transfectedcells that are administered to a host, preferably a human, are syngeneicor autologous (or autochthonous).

[0057] Cells transfected with nucleic acid encoding a SAg may alsoexpress a tumor associated antigen that is potentially present on hostcancer cells. For example, nucleic acid encoding a known tumor antigenare transfected into the SAg-containing cell, or a tumor cell thatendogenously contains many different tumor antigens are transfected withSAg-encoding nucleic acid. In the latter case, additional nucleic acidsencoding other polypeptides are transfected into the tumor cell. Forexample, nucleic acid encoding a carbohydrate modifying enzyme such asα1,3-galactosyltransferase, adhesion molecule, costimulatory moleculesuch as B7-1 and B7-2, MHC class I molecule and/or MHC class II moleculeare cotransfected into tumor cells together with nucleic acid encoding aSAg.

[0058] SAg-encoding nucleic acid can encode a mutant, variant, and/ormodified form of a SAg. These forms can be used to transfect T cells,alone or in combination with wild-type SAg-encoding acid. In addition,tumor cells are provided with the capacity to colonize sites ofmetastases and the ability to locally hydrolyze surrounding tumor groundsubstance and neovasculature by transfection of key bacterialStaphylococcal and Streptococcal enzymes, toxins and capsularpolysaccharides which confer upon the tumor cell additional tumorkilling properties and immunogenicity. The transfected genes includestaphylococcal hyaluronidase (tissue spreading factor), Staphylococcalerythrogenic toxin and Streptococcal capsular polysaccharide. The tumorcell may thus be capable of mimicking the tissue invasive anddestructive properties of the Streptococcus and Staphylococcus as theyproduce a sterile cellulitis localized to tumor sites.

[0059] These methods are used to treat any solid tumor such ascarcinoma, melanoma, and sarcoma, or cancers of hematopoietic originsuch as leukemia and lymphomas. This invention also provides for T cellsor NKT cells including γ/δT cells which after activation by SAgs innative or mutant form or transfected into tumor cells express surfacephenotypes which enhance their ability to traffic efficiently to tumorsites in vivo. Such phenotypes include CD44 and/or selective Vβexpression. In response to these SAg stimulants, the T cells produce TH1cytokines and, in particular, IFN-γ and IL-2.

[0060] Further, provided are methods of overcoming the T cellunresponsiveness of cancer patients by transfection of T cells fromtumor bearing host with the nucleic acids encoding the SAg receptor thusenabling these cells to be reactivated by exogenous SAg and used foradoptive immunotherapy in the same cancer patient.

[0061] Provided herein are SAg oligonucleotide andoligonucleotide-peptide compositions capable of targeting and deliveringSAgs to tumor sites in vivo without elimination by circulating naturallyoccurring SAg specific antibodies prevalent in the human cancerpatients. Provided also are compositions and methods for delivery oftherapeutic nucleic acid constructs to tumor sites in vivo usingtherapeutic genes carried by erythrocytes from patients with sickle cellanemia which have the unique capability of adhering to sites on tumorneovasculature.

1. Cancer

[0062] This invention is used to treat any type of cancer in a host atany stage of the disease. More particularly, the cancer is a solid tumorsuch as a carcinoma, melanoma, or sarcoma. This invention is used totreat cancers of hemopoietic origin such as leukemia or lymphoma, thatinvolve solid tumors. A host is any animal that develops cancer and hasan immune system such as mammals. Thus, humans are considered hostswithin the scope of the invention. Since the invention providesSAg-transfected cells as a vaccine, a cancer is one that a host islikely to develop based on family history or other criteria. In thiscase, the host is one that is susceptible to cancer.

2. Nucleic Acid

[0063] The term nucleic acid as used herein encompasses both RNA andDNA, including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand. The term isolated nucleic acidmeans that the nucleic acid is not immediately contiguous with both ofthe sequences with which it is immediately contiguous (one on the 5′ endand one on the 3′ end) in the naturally occurring genome of the organismfrom which it is derived. For example, an isolated nucleic acid moleculecan be, without limitation, a recombinant DNA molecule of any length,provided nucleic acid sequences normally found immediately flanking thatrecombinant DNA molecule in a naturally occurring genome are removed orabsent. Thus, an isolated nucleic acid molecule includes, withoutlimitation, a recombinant DNA that exists as a separate molecule (e.g.,a cDNA or a genomic DNA fragment produced by PCR or restrictionendonuclease treatment) independent of other sequences as well asrecombinant DNA that is incorporated into a vector, an autonomouslyreplicating plasmid, a virus (e.g., a retrovirus, adenovirus, or herpesvirus), or into the genomic DNA of a prokaryote or eukaryote. Inaddition, an isolated nucleic acid can include a recombinant DNAmolecule that is part of a hybrid or fusion nucleic acid sequence.

[0064] Typically, regulatory elements are nucleic acid sequences thatregulate the expression of other nucleic acid sequences at the level oftranscription and/or translation. Thus, regulatory elements include,without limitation, promoters, operators, enhancers, ribosome bindingsites, transcription termination sequences (i.e., a polyadenylationsignal), and the like. In addition, regulatory elements can be, withoutlimitation, synthetic DNA, genomic DNA, intron DNA, exon DNA, andnaturally-occurring DNA as well as non-naturally-occurring DNA. It isnoted that isolated nucleic acid molecules containing a regulatoryelement are not required to be DNA even though regulatory elements aretypically DNA sequences. For example, nucleic acid molecules other thanDNA, such as RNA or RNA/DNA hybrids, that produce or contain a DNAregulatory element are considered regulatory elements. Thus, recombinantretroviruses having an RNA sequence that produces a regulatory elementupon synthesis into DNA by reverse transcriptase are isolated nucleicacid molecules containing a regulatory element even though therecombinant retrovirus does not contain any DNA.

3. Transfection

[0065] The term “transfection,” of a nucleic acid into a cell, as usedherein is intended to include “transformation,” “transduction,” “genetransfer” and the like, as they are commonly used in the art.“Transfection ”is not intended to be limited to transfer of nucleic acidinto a cell by means of an infectious particle such as a retrovirus, asthe term may have been used originally. Rather any form of delivery andintroduction of a nucleic acid molecule, preferably DNA, into a cell,whether in the form of a plasmid, a virus, a liposome-based vector, orany other vector, so that the nucleic acid is expressed in the cell andits protein product(s) made, is included within the definition of“transfection.”

[0066] When a nucleic acid is said to “encode” a product other than aprotein, this language is intended to mean that it encodes the necessaryproteins/enzymes that are involved in, or required for, the synthesis ofthat product. For example, if a DNA molecule is said to encode LPS, itclearly encodes one or more proteins (enzymes) that are involved in thebiosynthesis of LPS. If a nucleic acid is said to “encode thebiosynthesis” of a structure, it means that the nucleic acid encodes theenzymes that participate in the creation of that structure. Inparticular for the carbohydrate structures referred to herein, thenucleic acids used in the invention are introduced into a cell thatnormally does not make, or makes little of, the carbohydrate structureso as to provide to that cell the genetic material for an enzyme orenzymes that generate the carbohydrate structure or modify a differentcarbohydrate structure to that one indicated. As a further example, DNAencoding a tumor antigen may directly encode a protein/peptide tumorantigen, or alternatively, may encode proteins or peptides that eithercontribute structurally to, or catalyze the synthesis of, a tumorantigen which is partly protein (e.g., lipoprotein or proteoglycan) ortotally non-protein (e.g., a glycolipid).

[0067] The invention provides methods of treating cancer in a host bytransfecting cells with SAg-encoding nucleic acid. Suitable host ornon-host cells for transfection include, without limitation, endothelialcells, DCs, monocytes, macrophages, B cells, T cells, immunocytes,muscle cells, fibroblasts, NK cells, NKT cells (TCR αβ⁺ CD4^(neg) andCD8^(neg)), γ/δ T cells and tumor cells. The terms accessory cell andantigen presenting cell (APC) can be used interchangeably and includecells having the ability to process and present antigens to T cells aswell as to provide both defined and less well defined growth anddifferentiation factors (costimulatory signals) during an ongoing immuneresponse.

[0068] Cells are transfected in vivo or in vitro. When transfected invivo, the cells are of host origin. When transfected in vitro, the cellsare autologous, allogeneic, or xenogeneic to the host to provideadditional immunogenicity. In addition to being transfected with nucleicacid encoding a SAg, the cells are transfected with nucleic acidencoding any other polypeptide including, without limitation, agalactosyltransferase, staphylococcal hyaluronidase and/or erythrogenictoxin, streptococcal capsular polysaccharide, CD44, tumor antigen,costimulatory molecule such as B7-1 and B7-2, adhesion molecules, MHCclass I molecule and/or MIIC class II molecule. Nucleic acids encodingthe molecules are cotransfected with the SAgs. But for others, includingbut not limited to Staphylococcal hyaluronidase, erythrogenic toxin,Streptococcal capsular polysaccharide and CD44 genes, the nucleic acidsencoding the SAgs are fused to other nucleic acids resulting inexpression of a fusion protein.

[0069] Methods for in vivo and in vitro transfection of cells are wellknown. For example, two books in the series Methods in MolecularMedicine published by Humana Press, Totowa, N.J., describe in vivo andin vitro transfection protocols that are adaptable to the presentinvention (Vaccine Protocols edited by Robinson et al., (1996) in GeneTherapy Protocols edited by Robbins et al., Humana Press, Totowa, N.J.(1997)). Transfection protocols are also discussed elsewhere ((Sambrook,J. et al., Molecular Cloning, Second Edition, Cold Springs HarborLaboratory Press, Plainview, N.Y., (1989)). In addition, use of variousvectors to target epithelial cells, use of liposomal constructs, methodsof transferring nucleic acid directly into T cells, hematopoietic stemcells, and fibroblasts, methods of particle-mediated nucleic acidtransfer to skin cells, and methods of liposome-mediated nucleic acidtransfer to tumor cells have been described elsewhere. (Feigner, PL etal., Cationic Lipids for Intracellular Delivery of Biologically ActiveMolecules, U.S. Pat. No. 5,459,127, issued Oct. 17, 1995; Feigner, PL,Cationic Lipids for Intracellular Delivery of Biologically ActiveMolecules, U.S. Pat. No. 5,264,618, issued Nov. 23, 1993; Felgner, PL,Exogenous DNA Sequences in a Mammal, U.S. Pat. No. 5,580,859 issued Dec.3, 1996; Felgner, PL, A Protective Immune Response in a Mammal byInjecting a DNA Sequence, U.S. Pat. No. 5,589466 issued Dec. 31, 1996).

[0070] Further, use of ligand-based nucleic acid carriers to effecttransfection of mammalian cells in vivo has been described elsewhere (Wuet al., J. Biol. Chem., 262:4429-4432 (1987); Wu et al., J. Biol. Chem.,263:14621-14624 (1988); Wu et al., J. Biol. Chem., 264:16 985-16987(1989); Wu et al., J. Biol. Chem., 266:14338-14342 (1991); and GarriguesJ et al., Am. J. Path. 142:607-622 (1993)). Briefly, plasmid DNA isconjugated to a desialylated branched carbohydrates such as orosomucoidby carbodiimide crosslinking to polylysine and targeted to asialoproteinreceptors expressed on cells in the liver. In addition, enhanced nucleicacid delivery and expression can be achieved using a ligand-polylysineconjugate coupled to carbohydrate moieties on viruses that is thencombined with DNA. These preparations are suitable for parenteralinjection and are readily taken up by cells expressing asialoproteinreceptors in the liver after which the DNA is internalized andintegrated into the host genome. In addition, nucleic acid can beadministered intravenously, intramuscularly, or subcutaneously to inducea response in a host.

[0071] Thus, targeting nucleic acid to the surface of particular cellsis accomplished by conjugating nucleic acid to molecules that bind to acell surface structure such as a receptor. Examples of cell surfacestructures that can be targeted include, without limitation, thetransferrin receptor, and asialoglycoprotein receptor. The moleculesthat bind cell surface structures and are conjugated to nucleic acid fortargeting can be, without limitation, natural ligands for the surfacestructure, synthetic compositions that exhibit specific binding, andantibodies directed against the surface structure. For example, amonoclonal antibody specific for a cell surface epitope such as the BR96antibody that recognizes Le^(x) carbohydrate epitope abundantlyexpressed by colon, breast, ovary, and lung carcinomas can be used.Other monoclonal antibodies can include, without limitation, those thatrecognize growth factor receptors, transferrin receptors, IL-2receptors, epidermal growth factor receptors, the hev oncogene, andTAPA-1 as well as any other antibody having specificity for a surfacestructure that can be internalized.

[0072] Liposomes containing nucleic acid are also targeted to specificcell types such that the nucleic acid is expressed. For example, nucleicacid is loaded into or attached to cationic DOTMA:doleoylphosphatidylethanolamine (DOPE) liposomes that contain exposedmolecules that bind to a cell surface structure such as tumor cells ortumor microvasculature (Example 5). The molecules that bind cell surfacestructures and are attached to liposomes can be, without limitation,natural ligands for the surface structure, synthetic compositions thatexhibit specific binding, and antibodies directed against the surfacestructure. Maximal transfer of nucleic acids encoding SAgs is attainedby synthesizing the liposomes with an appropriate ratio of nucleic acidto lipid. In addition, these nucleic acid-containing liposomes areadministered intravenously, intramuscularly, or subcutaneously to inducea response in a host.

[0073] Naked nucleic acid is also administered to a host. For example,naked pharmaceutical-grade plasmid DNA are injected into a hostintramuscularly such that it is expressed by host cells (U.S. Pat.Nos.5,589,466; 5,580,599; 5,264,618; 5,459,127; and 5,561,064). Inaddition, cationic lipids are used to deliver biologically activemolecules, such as oligonucleotides to host cells in vivo (U.S. Pat.Nos. 5,264,618, 5,459,127, and 5,561,064). Thus, nucleic acid encoding aSAg is administered to a host in naked or cationic lipid form such thatthe SAg is expressed. It is noted that any nucleic acid described hereincan be administered in vivo as naked DNA. Further, other methods ofadministering naked DNA to a host can be used such as those related tothe direct injection of naked DNA for use in vaccines (Cohen et al.,Science 259:1691-1692 (1993); Corr et al., J. Exp. Med. 184:1555-1560(1996); Varmus et al., Proc. Natl. Acad. Sci. USA 81:5849-5852 (1984);and Benveniste et al., Proc. Natl. Acad. Sci. USA 83:9551-9555 (1986)).

[0074] Our previous patent applications which are hereby incorporated byreference include U.S. patent application Ser. No. 07/416,530, filedOct. 3, 1989, U.S. patent application Ser. No. 07/466,577, filed Jan.17, 1990, U.S. patent application Ser. 07,891,718, filed Jun. 1, 1992,U.S. patent application Ser. 08/025,144, filed Mar. 2, 1993, U.S. patentapplication Ser. 08/189,424, filed Jan. 31, 1994, U.S. patentapplication Ser. 08/491,746, filed Jun. 19, 1995, PCT applicationsPCT/US91/00342, and PCT/US94/02339. These applications have givencomprehensive description of the SAg genes, the creation of highenterotoxin producing mutant strains as well as recombinant methods ofproduction of SAgs. In addition, methods of treating cancer bytransfecting tumor cells in vivo and in vitro with SAg nucleotides usingwell defined recombinant technology have been described in theseapplications. Subsequently, Dow et al., (J. Clin. Invest. 99: 2616-2624(1997)) described in vitro and in vivo transfection of eukaryotic cellswith SAg DNA which was capable of inducing inflammatory responses invivo. It is noted that the SAg genes have been cloned and theirsequences delineated before 1988 and methods used to transfect cells invivo or in vitro with nucleic acids encoding polypeptides are also wellknown in the art.

4. Constructs

[0075] Tumor cells are transfected with various nucleic acids which aredesigned to increase their immunogenicity and to provide them withcapacity to traffic to metastatic sites where they may initiate a potentinflammatory and immune response. Such constructs of this invention canbe linear or circular nucleic acids obtained from mammals or bacteriathat encode a polypeptide such as a SAg, mutant SAg, erythrogenic toxin,enzymes involved in the biosynthesis of glycosyltransferases, bacterialglycosylceramides, LPS's, lipoproteins, capsular or membranepolysaccharides, microbial toxins and enzymes such as hyaluronidase,collagenase, elastase, coagulase, protease, kinase, lipase. Constructsmay also contain tumor associated antigens, costimulatory molecules suchas B7-1 and B7-2, adhesion molecules, receptor molecules such as SAgreceptors, CD1, CD14, MHC class I molecules and/or MHC class IIreceptors. Such constructs may also contain amplified nucleic acidsassociated with tumors such as oncogenes, transcription factors,angiogenesis factors and receptors, tumor growth factor receptors,chimeric receptors. The latter nucleic acids may be linked toSAg-encoding nucleic acid to produce heightened expression of the SAg.The amplified nucleic acids may include tumor tissue specific promotersand nucleic acids that direct the colonization or metastasis of tumorsto selected sites in vivo.

[0076] Constructs can also contain elements that regulate and/or promotethe expression of an encoded polypeptide. For example, a constructcontaining nucleic acid that encodes enterotoxin B (SEB) can have astrong promoter element upstream of the SEB encoding sequence. Inaddition, constructs can contain nucleic acid that anchors an encodedpolypeptide to the cell surface after expression. For example, aconstruct containing nucleic acid that encodes SEB can contain amembrane-anchoring sequence such as nucleic acid that encodes ahydrophobic stretch of amino acids or a glycosylphosphatidylinositol(GPI)-anchoring motif. Thus, the SAg, or other polypeptides as well, canbe anchored in the plasma membrane by coupling to membrane lipids orglycolipids. These anchors can be attached to the C terminus of thepolypeptide in the endoplasmic reticulum. Alternatively, a SAg known tobe associated with the cell surface after expression can be used such asthe mammary tumor viral (MMTV) SAg that is GPI-linked.

[0077] In one embodiment, SAgs as well as SAg receptors are engineeredto remain anchored to the surface of transfected cells when the cell isto be used for immunization. Likewise, when a SAg receptor gene istransfected into anergized T cells from cancer patients, it is desirableto express the receptor on the cell surface so that they are readilyrecognized and activated by exogenous receptor bound SAg. In contrast,when it is desirable to use SAg transfected cells to activate T cells invivo or ex vivo or to promote trafficking of transfected tumor cells tometastatic sites in vivo, it is suitable for the SAg to be secreted fromthe transfected cells.

[0078] In additional embodiments, potent tumor specific effector T orNKT cell clones are produced with overexpressed Vβ regions of their TCRsmaking them highly receptive to activation by exogenous SAg. LikewiseCD44 genes are transfected into T cells or NKT cells making them moresusceptible to expression of this epitope after SAg stimulation.

[0079] Constructs also contain a selectable marker or reporter such thattransfected cells can be isolated. For example, a construct containingnucleic acid that encodes a SAg can also contain nucleic acid thatencodes a polypeptide that confers resistance to a selection agent suchas neomycin (also called G418), puromycin, or kanamycin.

[0080] Nucleic acid and nucleic acid constructs of the present inventionare incorporated into a vector, an autonomously replicating plasmid, ora virus (e.g., a retrovirus, adenovirus, or herpes virus). Typically,these vectors, plasmids, and viruses can replicate and functionindependently of the cell genome or integrate into the genome. Vector,plasmid, and virus design depends on, for example, the intended use aswell as the type of cell transfected. Appropriate design of a vector,plasmid, or virus for a particular use and cell type is within the levelof skill in the art. In addition, a single vector, plasmid, or virus canbe used to express either a single polypeptide or multiple polypeptides.It follows that a vector, plasmid, or virus that is intended to expressmultiple polypeptides will contain one or more operably linkedregulatory elements capable of effecting and/or enhancing the expressionof each encoded polypeptide.

[0081] The term “operably linked” means that two nucleic acid sequencesare in a functional relationship with one another. For example, apromoter (or enhancer) is operably linked to a coding sequence if iteffects (or enhances) the transcription of the coding sequence. Aribosome binding site is operably linked to a coding sequence if it ispositioned to facilitate translation. Operably linked nucleic acidsequences are often contiguous, but this is not a requirement. Forexample, enhancers need not be contiguous with a coding sequence toenhance transcription of the coding sequence.

[0082] A vector, plasmid, or virus that directs the expression of apolypeptide such as a SAg can include other nucleic acid sequences suchas, for example, nucleic acid sequences that encode a signal sequence oran amplifiable gene. Signal sequences are well known in the art and canbe selected and operatively linked to a polypeptide encoding sequencesuch that the signal sequence directs the secretion of the polypeptidefrom a cell. An amplifiable gene (e.g., the dihydrofolate reductase[DHFR] gene) in an expression vector can allow for selection of hostcells containing multiple copies of the transfected nucleic acid.

[0083] Standard molecular biology techniques are used to construct,propagate, and express the nucleic acid, nucleic acid constructs,vectors, plasmids, and viruses of the invention ((Sambrook, J. et al.,supra; Maniatis et al., Molecular Cloning (1988); and U.S. Pat. No.5,364,934. For example, prokaryotic cells (e.g., E. coli, Bacillus,Pseudomonas, and other bacteria), yeast, fungal cells, insect cells,plant cells, phage, and higher eukaryotic cells such as Chinese hamsterovary cells, COS cells, and other mammalian cells can be used.

[0084] Constructs are used in vivo or ex vivo or in combination as inExample 5-7, 16-23. They are used to immunize a host by direct in vivoadministration or they are used ex vivo to activate T cells or NKT cellsto become tumor specific effector cells which are employed for adoptiveimmunotherapy of cancer by methods and models (Examples 7, 16, 19-23).

[0085] To test the anti-tumor-inducing ability of a particular constructas well as the transfected cell itself, the following general assay isperformed. B16 melanoma, A20 lymphoma, host tumor cells, or any othertumor cell lines appropriate to the host (i.e., having tumor antigensexpected to be present on the host tumor cells) are transfected with agiven construct. Appropriate numbers of transfected cells (e.g., 10⁵-10⁷cells) are then implanted subcutaneously into animals such as mice,rats, rabbits, or the like and 1-6 months later untransfected tumorcells are implanted. Tumor outgrowth from the untransfected tumor cellsis measured and compared to control animals not given the transfectedtumor cells. If tumor outgrowth is reduced or prevented, then thetransfected cells are effective anti-tumor agents useful as tumorvaccines. Alternatively, 10⁵-10⁷ transfected tumor cells can be given3-10 days after the appearance of established tumors from untransfectedtumor cells. If tumor outgrowth is reduced or arrested, then thetransfected cells are effective anti-tumor agents useful in treatingestablished tumors.

[0086] To test the anti-tumor effect of SAg activated T cells, γ/δ Tcells NKT cells or T cells clones overexpressing Vβ or CD44, thefollowing general protocol is used. Lymph node cells from C57/B1 micebearing MCA 205 or 207 sarcomas which were implanted in the adjacentinguinal region three to ten days before are extracted and placed intissue culture. The cells are incubated with various enterotoxins fortwo days and then with IL-2 for an additional two to three days. Thecells are then harvested and injected into syngeneic mice withestablished pulmonary metastases (six to twelve days after tumorinjection). Three weeks later the animals are evaluated for pulmonarymetastases compared to controls which receive no cells or cells thatwere stimulated without enterotoxins. The adoptively transferred cellsmay be enriched for NKT cells or γ/δ T cells or α/β+ T cells alone whichare selectively injected into tumor bearing hosts. Likewise, they areselected for predominant expression of the CD44 phenotype during the SAgactivation phase at which time the CD44 enriched population is harvestedand used for adoptive immunotherapy. The dose of injected T cells, NKTcells or γ/δ T cells and/or CD44 enriched cells (which are produced byany of these T cell, NKT cell or γ/δ T cell populations) range from 10⁶to ⁷ and are be given on a schedule of once weekly for one to fourweeks.

5. Superantigens (SAgs)

[0087] SAgs are polypeptides that have the ability to stimulate largesubsets of T cells. SAgs include Staphylococcal enterotoxins,Streptococcal pyrogenic exotoxins, Mycoplasma antigens, rabies antigens,mycobacteria antigens, EB viral antigens, minor lymphocyte stimulatingantigen, mammary tumor virus antigen, heat shock proteins, stresspeptides, clostridial and toxoplasmosis antigens and the like. Any SAgcan be used as described herein, although, Staphylococcal enterotoxinssuch as SEA, SEB, SEC, and SED and streptococcal pyrogenic exotoxinssuch as toxic shock-associated toxin (TSST-1 also called SEF) arepreferred.

[0088] When using enterotoxins, the region related to emetic activitycan be omitted to minimize toxicity. In addition, SAgs can bederivatized to minimize toxicity. The level of toxicity may not be aconcern when using SAg transfected cells to activate lymphocytes ex vivosince the lymphocytes can be rinsed of SAg polypeptide prior toadministration to a host.

[0089] The nucleic acid sequences that encode SAgs are known and readilyavailable. For example, Staphylococcal enterotoxin A (SEA), SEB, SEC,SED, SEE, TSST-1, and Streptococcal pyrogenic exotoxin (SPEA) have beencloned and can be expressed in E. coli (Betley M J and J J Mekalonos, J.Bacteriol. 170:34 (1987); Huang I Y et al., J. Biol. Chem., 262:7006(1987); Betley M et al., Proc. Natl. Acad. Sci. USA, 81:5179 (1984);Gaskill M E and S A Khan, J. Biol. Chem., 263:6276 (1988); Jones C L andS A Khan, J. Bacteriol., 166:29 (1986); Huang I Y and M S Bergdoll, J.Biol. Chem., 245:3518 (1970); Ranelli D M et al., Proc. Nat. Acad. Sci.USA 82:5850 (1985); Bohach G A, Infect Immun., 55:428 (1987); Bohach GA, Mol. Gen. Genet. 209:15 (1987); Couch J L et al., J. Bacteriol.170:2954 (1988); Kreiswierth B N et al., Nature, 305:709 (1983); CooneyJ et al., J. Gen. Microbiol., 134:2179 (1988); Iandolo J J, Annu. Rev.Microbiol., 43:375 (1989); and U.S. Pat. No. 5,705,151)). Additionalnucleic acid sequences encoding SAgs are described elsewhere (Bohach etal., Crit. Rev. in Microbiology 17:251-272 (1990); (Kotzin, B L et al.,Adv Immunol 54: 99-165 (1993))

[0090] PCR can be used to isolate SAg-encoding acid. For example, thenucleic acid encoding SEA, SEB, and TSST-1 can be isolated as describedelsewhere (Dow et al., J. Clin. Invest. 99:2616-2624 (1997)). Briefly,the following primers can be used to amplify the SAg-encoding nucleicacid:

[0091] SEA forward: GGGAATTCCATGGAGAGTCAACCAG,

[0092] SEA backward: GCAAGCTTAACTTGTTAATAG;

[0093] SEB forward: GGGAATTCCATGG-AGAAAAGCG,

[0094] SEB backward: GCGGATCCTCACTTTTTCTTTG; and

[0095] TSST-1 forward:GGGGTACCCCGAAGGAGGAAAAAAAAATGTCTACAAACGATAATATAAAG,

[0096] TSST-1 backward: TGCTCTAGAGCATTAATTAATTTCTGCTTCTATAGTTTTTAT.

[0097] The full-length TSST-1 nucleic acid sequence is cloned into aeukaryotic expression vector (pCR3; In Vitrogen Corp., San Diego,Calif.), whereas only the sequence corresponding to the mature SEB andSEA (sequences minus the putative bacterial signal sequences) is clonedinto pCR3. Removal of the SEB and SEA signal sequences increases thelevel of expression in transfected cells. The plasmids are grown inEscherichia coli and plasmid DNA extracted by the modified alkalinelysis method and purified on a CsC1 gradient.

[0098] Nucleic acids encoding mutant or variant SAgs are also considerednucleic acid sequences encoding SAgs within the scope of the invention.For example, a mutant SAg-encoding acid sequence is engineered such thatthe resulting SAg is devoid of amino acid residues, e.g., histidine,known to produce toxicity. Likewise, SAg-encoding nucleic acid isengineered to contain or lack sequences that facilitate the selectivebinding of SAgs to certain Vβ regions of the TCR present on T cells orto ganglioside, mannose (or other carbohydrate) receptor, certainregions of MHC class II, and/or enterotoxin receptors present on tumorcells, antigen presenting cells (APCs), and/or lymphocytes.

[0099] Nucleic acid sequences that encode a SAg are also fused, inframe, with nucleic acid that encodes another polypeptide. This largernucleic acid is termed herein a SAg fusion gene and the resultingpolypeptide product is a SAg fusion product. Nucleic acid sequences thatare fused to SAg-encoding nucleic acid include, without limitation,nucleic acid sequences that encode tumor antigens, costimulatorymolecules, adhesion molecules and MHC class II molecules. Thesuperantigen fusion product is secreted by a transfected cell, expressedon the cell surface or it may remain intracellular in nucleic acid orpartly processed form.

[0100] SAgs are also isolated and purified from their natural source aswell as from a heterologous expression system such as E. coli. Likewise,SAg-containing polypeptides (e.g., SAg fusion products) are isolated andpurified from a heterologous expression system. In addition,Staphylococcus strains producing high levels of enterotoxin have beenidentified and are available. For example, exposingenterotoxin-producing Staphylococcus aureus to mutagenic agents such asN-methyl-N-nitro-N-nitrosoguanidine results in a 20 fold increase inenterotoxin production over the amounts produced by the parent wild-typeStaphylococcus aureus strain (Freedman M A and Howard M B J. Bacteriol.,106:289(1971)).

6. Glycosylated SAgs and SAgs Conjugated to Glycosylceramides,Lipopolysaccharides, Glycans and Lipoarabinomannans: Presentation on CD1Receptors for Activation of T or NKT Cells and Differentiation to TumorSpecific Effector Cells

[0101] In a tumor cell or accessory cell, nucleic acid signal sequencesare integrated into nucleic acids encoding the SAg molecules in order toroute them to the Golgi apparatus and endoplasmic reticulum of tumorcells where they are glycosylated via appropriate glycosyltransferases(precedents from the selective transferases used to producemonogalactosylceramide in the Sphingomonas paucimobilis) to produce aproteoglycan with structural similarity to LPS, lipoteichoic acid,GalCer, α-gal, Streptococcus capsular polysaccharide. This construct isthen secreted as an immunogenic “ground substance.” Alternatively, theresulting SAg glycolipid is anchored to the membrane, expressed on thecell surface and routed specifically to CD1 receptors.

[0102] SAgs which are glycosylated by the above intracellular processeshave improved capacity to bind surface structures such as mannosereceptors, ganglioside receptors and CD1 receptors. Generally, thenucleic acids encoding a SAg are modified to include a signal sequencefor routing to the Golgi apparatus and a core sequence which initiatesglycosylation. It is important that the V Vβ TCR binding region is notblocked by the added carbohydrate modifications. For example, anN-linked glycosylation site (in the sequence Asn X Ser/Thr where X isany residue except Pro) is engineered into SAg-encoding acid sequenceswhich do not functionally interfere with TCR binding and activation. Thenucleic acid encoding these signal sequences and core bindingglycosylation sites of SAgs are fused to nucleic acids encoding SAg andthe fusion gene used to transfect tumor cells of a host. In addition,glycosylated forms of SAgs are expressed in a heterologous eukaryoticexpression system such as yeast cells or baculovirus-infected insectcells. In gram negative bacteria (such as E. coli), nucleic acidsencoding SAgs are fused to nucleic acids encoding LPS's, in grampositive bacteria (such as Staphylococcus or Streptococcus), to nucleicacids encoding capsular polysaccharides and teichoic acids and inmycobacterial species to nucleic acids encoding lipoarabinan.

[0103] The gram negative bacterium Sphingomonas paucimobilis producesthe monogalactosylceramide. In this bacterium, nucleic acids encodingSAgs (containing serine) are fused to nucleic acids encoding anddirecting the synthesis of glycosylceramides and monogalactosylceramidein particular. The resulting galactosylceramide-SAgs are powerful T cellstimulants. The same procedure is followed in bacteria which naturallyproduce LPS's such as E. coli, Salmonella or Klebsiella or for bacteriawhich naturally produce lipoarabinomannans glycans or polysaccharidescontaining cell walls such as Mycobacterium and Streptococcusrespectively. The SAg-polysaccharide constructs bind to CD1 receptors ofantigen presenting cells. They are then capable of activating NKT cellseither in vivo or ex vivo to become tumor specific effector cells inresponse to IL-12.

[0104] SAgs are also conjugated genetically or biochemically as inExample 5 to LPS's via a natural high affinity binding site for LPSbinding protein (LPB). Once bound, the SAg catalyzes the binding of LPSmonomers to CD14 and CD1 receptors in a fashion similar to that of LPB.In this way, the conjugates are capable of activating T cells for use invivo or ex vivo for adoptive immunotherapy while preserving theanti-apoptotic effect of LPS on SAg activated T cells. Examples of theirpreparation and use in vivo and in vitro are given in Examples 4, 7, 15,16, 18-23.

[0105] In addition, SAgs similarly conjugated to lipoarabinomannans andglycans are integrated into lymphomonocytic cell membranes viaglycosylphosphatidylinositol anchors. These SAg-lipoarabinomannancomplexes are expressed or secreted by antigen presenting cells or tumorcells. They are also bound to CD1, mannose or class II receptors inwhich form they are used to activate T or NKT cells. These constructsare administered in vivo or they are used ex vivo to produce tumorspecific effector cell populations (T cell or NKT cells) which areemployed for adoptive immunotherapy of cancer (Examples 5, 15-16,18-23).

[0106] Mannose receptor expression is upregulated by cytokines. Forexample, accessory cells including DCs, and tumor cells express mannosereceptors on their surfaces after GM-CSF treatment. SAgs are bound tomannose receptors by transfecting cells with nucleic acids encoding SAgwhich also consist of nucleic acids encoding signal sequences andglycosylation sites which, in the presence of appropriateglycosyltransferases, produce mannosylated SAgs. These preferentiallybind to mannose receptors. In addition, glycosylated SAgs bind toamphipathic cell surface gangliosides and glycolipids via hydrophobicinteractions. These glycosylated SAgs presented in a form bound tomannose receptors are capable of activating α/β cells, γ/δ T cells, NKcells, and NKT cell populations. They are used either in vivo by directadministration or ex vivo to produce a tumor specific effector cellpopulation (T cell, NK or NKT cells) for use in adoptive immunotherapyof cancer (Examples 4, 5, 15, 16, 18-23).

7. SAgs Conjugated to Glycosylceramides Gangliosides and Verotoxins (VT)

[0107] Amphipathic gangliosides bound to tumor cell surfaces such asGD1, GD2, GD3, GM1, GM2, GM3, GQ1 and GT1 are capable of bindingexogenous SAgs. The binding of a SAg to the surface of a tumor cellcreates an immunogen on the tumor cell surface. Tumor cells transfectedwith nucleic acids encoding glycosyltransferases overexpressgangliosides, producing a greater surface density of gangliosidemoieties available to bind exogenous SAgs. Enterotoxins bind to cellsurface amphipathic gangliosides and/or glycophorins via theirhydrophobic residues while preserving their T cell binding properties.SAgs are also glycosylated intracellularly by addition of aglycosylation site or by chemical conjugation of a carbohydrate moietyusing methods well described in the art. In glycosylated or native form,the SAgs bind to surface ganglioside while retaining their T cellactivating properties. Overexpression of the hydrophobic regions of themolecule promotes binding to the surface gangliosides (Example 5).

[0108] Examples from nature of exogenous proteins that bind to cellsurface gangliosides include falciparum malarial merozoite whichcombines with gangliosides associated with the Duffy blood group andinduce long standing and durable protection and tetanus toxin whichbinds to surface gangliosides with highest affinity for the disialylgroups linked to inner galactosyl residues.

[0109] Enterotoxin B contains a T cell activating sequence which ischemically cross-linked or polymerized using bifunctional agents such ascarbodiimide, glutaraldehyde or formaldehyde by established methods wellknown in the art. These polymers are then bound to gangliosidesexpressed on tumor cells such as GD1, GD2, GQ1,GD3 or GM1, GM2, GM3,GT1. In monomeric or polymerized form, SAgs also bind tomonogalactosylceramides which are free or bound to CD1 receptors ontumor cells or antigen presenting cells via hydrophobic interactions.The monogalactosylceramide binds to hydrophobic sequences on the SAgwhich are expressed at multiple sites on the molecule. In oneembodiment, the lauroyl group [CH₃(CH)₁₀CO] or the group [CH₃(CH)₁₃CO]is covalently added to each of the peptide's amino terminus to serve asa of the CD1 receptor. The key SAg peptide sequence such as of SEB(amino acids 225-234) which confers T cell activating properties istandemly repeated to various lengths prior to lipid conjugation.

[0110] Hydrophobic SAg peptides(such as Trp, Tyr, Phe, Leu, and Ile) arescreened for binding to glycosylceramides immobilized on CD1 receptorsor via adsorption chromatography with immobilized glycosylceramide. TheSAg sequences with the greatest affinity for the CD1 receptor areselected for conjugation to the glycosylceramides and LPS's.Alternatively, the SAg sequence is screened for affinity for the CD1 orMHC class II receptor using a peptide phage display library as describedin Examples 4. Likewise, pre-formed SAg-glycosylceramide or LPScomplexes are also screened for affinity for the CD1 or MHC class IIreceptor (Example 4). These lipopeptide complexes are then screened forT cell proliferative activity and IL-12 production. The monomeric orpolymerized SAg in native or glycosylated form binds to themonoglycosylceramides or gangliosides expressed on CD1 receptors on thetumor cell surface.

[0111] Therapeutic Construct: SAg-Glycosylceramide Conjugates

[0112] SAgs have an affinity for glycosphingolipids especially thosewith terminal or subterminal Gal(α1-4)Gal residues. Such residues areexpressed on tumor cells as Gal(α1-4)Gal(β1-4)GlcCeramide(globotriaosylceramide or Gb3) and Gal(α1-4)GalCeramide(galabiosylceramide or Gb2). Gb3 and Gb2 also known as CD77, Burkitt'slymphoma antigen, and the human blood group p^(k) antigen are thenatural receptors for Shiga toxins and VT's. Shiga toxin, a 69-kDacomplex of proteins comprised of five B-subunits (7 kDa each) and oneA-subunit (30 kDa) has high affinity for the terminal digalactose of Gb3or Gb2. Methods for their preparation and isolation are described inExample 41. Once bound to the tumor cell, these toxins are internalizedand induce apoptosis.

[0113] The synthetic pathway for neutral glycosphingolipids ineukaryotic cells is known. Glucosylceramide (GlcCer) is the precursor oflactosylceramide (LacCer), which leads, in order, to Gb3 andglobotetraosylceramide (Gb4). Different Golgi enzymes are responsiblefor addition of monosaccharides from nucleotide-sugar donors in eachstep of the pathway. Globotriaosylceramide synthase(UDP-galactose:lactosylceramide α 1-4-galactosyltransferase) has beenpurified. In the cytoplasm, the A-subunit of the Shiga toxin or VT isprocessed by a trypsin-like cleavage. The “activated” 27-kDa A-subunitinactivates 60S ribosomes by depurination of a single nucleotide in 28SrRNA, rendering ribosomes incapable of carrying out peptide elongation.

[0114] The present invention provides therapeutically active solublecomplexes comprising SAg and glycosphingolipids which have terminal orsubterminal Gal(α1-4)Gal residues and Shiga toxin receptors Gb3 and Gb2,(collectively referred to as “GTSG1-4”). These complexes include but arenot limited to SAg-GP1-GTSG1-4 complexes, and synthetic and functionalderivatives thereof. Such structures appear naturally on surfaces ofcertain tumor cells such as astrocytoma, Burkitt's lymphoma and ovariancarcinoma. Methods of preparing and isolating glycosylceramides and VTsare given in Examples 41 and 55.

[0115] SAgs also have a demonstrable affinity for galactosylceramidescontaining Gal(α1-4)Gal residues. Methods of assessing SAg binding toGTSG1-4 are provided given in Example 43. These conjugates are also shedfrom SAg-transfected tumor cells as binary complexes of SAg-GTSG1-4 orternary complexes of SAg-GPI-GTSG1-4, in free form, as vesicles or asexosomes(see Sections 38 and Example 38). Methods of isolating andcharacterizing these shed complexes appear in Section 38 and Example 42.The complexes may also be prepared by chemical or genetic methods(Example 5). SAg-GTSG1-4 or SAg-GPI-GTSG1-4 complexes or exosomes areuseful as a preventative vaccine or against established tumor. They arealso useful in vivo by direct administration or ex vivo where they areloaded onto antigen presenting cells comprising CD1 or MHC receptors toactivate NKT and T cells to produce tumor specific effector T or NKTcells for adoptive therapy of cancer (Examples 5, 7, 14, 15, 16, 18-23,38).

[0116] Therapeutic Construct: Tumor Cells Expressing SAgs andGalactosylsylceramides

[0117] Additional immunogenic complexes comprising SAgs bound to tumorcells, DCs DC/tc constructs expressing surface Gb2 and Gb3 or otherglycosphingolipids containing terminal Gal(α1-4)Gal are prepared bytransfecting these cells with nucleic acids encoding a SAg. Thetransfected cell expresses the SAg in the context of theglycosphingolipid comprising the terminal or subterminal Gal(α1-4)Galmoiety. Alternatively, free or GPI linked glycolipids containing SAgpeptides or polypeptides bind to tumor cells or accessory cells intissue culture (Section 38). The expression of Gb3 and Gb2 on tumorcells is optionally upregulated by various cytokines, including IFNα andTNFα, before contacting the SAg

[0118] Tumor cells, accessory cells or fused tumor/accessory cellstransfected with SAg which are not naturally endowed with the GalCer(optionally coupled to SAg) acquire these molecules in free orGPI-linked form from surrounding media or by transfer from liposomes orvesicles (exosomes) which express them (Section 38 and Example 5). Theresulting cells, coexpress SAgs and glycosylceramides or otherglycosylceramides capable of stimulating an effective T or NKT cellimmune response. Multidrug resistant (MDR) tumor cells or cell lineswhich naturally accumulate and express intracellular glycosylceramidesare useful in this invention. MDR agonists such as SDA PSC 833, acyclosporin analogue, and fumonisin B1, a ceramide synthase inhibitor,are employed to induce ceramide accumulation in MDR cells (Example 45).Tumor cells or accessory cells which overexpress key glycosylceramidesdue to transfection with a1-2, α1-4, α1-6 glycosyltransferases (Example38) or a natural or induced deficiency of (α-galactosidase are alsouseful. In addition, tumor cells with high concentrations of GalCerexpressed on their surface or that of accessory cells are generated byincubation with ceramides containing a 2-hydroxy fatty acid C6OH. Tumorcells selectively convert them to GalCer, galabiosylceramide andsulfatide in the trans-Golgi network where they are sorted andtransported selectively to the cell surface. Methods for this selectivebiosynthesis of GalCer with hydroxy fatty acids are in Example 46.

[0119] These fused SAg-tumor cell/accessory cell constructs are used toactivate a T or NKT cell population. They are used in vivo by directadministration or ex vivo to produce a population of tumor specificeffector cells (T cells or NKT cells) for adoptive therapy of cancer(Examples 5, 7, 14, 15, 16, 18-23, 38).

[0120] SAg-VT Conjugates to Induce Tumor Cell Apoptosis

[0121] The present invention contemplates the induction of apoptosis intumor cells expressing Gb2 and Gb3 (or other glycosphingolipidscontaining terminal Gal(α1-4)Gal) by using free SAgs, conjugates andfused DNA that comprises SAg, SAg peptide or SAg-encoding DNA fused tointact VT or to VT A or B chains. Preparation of these conjugates andfusion proteins from their corresponding DNA, polypeptides or functionalderivatives is provided in Examples 1 and 5. These conjugates induceapoptosis by binding to tumor cell glycosphingolipid receptors havingterminal Gal(α1-4)Gal. Methods of assessing tumor cell apoptosis are inExample 44. CD19 or IFN-αpeptide sequences and generic carbohydraterecognition domains which bind Gal(α1-4)Gal structures are also useful.CD19, a B-cell restricted differentiation antigen, naturally binds toGb3 and Gb2 on the cell surface which includes apoptosis. CD19 hasVT-like sequences in the N-terminal extracellular domain (NBRF proteindata bank) that have 41%, 34% and 37% sequence identity to VT1, VT2, andVT2e B subunits, respectively. When compared to a consensus VT Bsequence, the CD19 sequences show 49% identity. Binding of these peptidesequences to membrane Gal(α1-4)Gal-containing glycolipids facilitatesreceptor mediated induction of apoptosis.

[0122] The IFN-α receptor has a 63-kDa extracellular peptide withregions of amino acid identity to domains in the VT B subunit implicatedas Gb2/Gb3 binding sites.

[0123] The preferred targets of the above conjugates on tumor cells arethe naturally expressed Shiga toxin receptors Gb3 and Gb2 with aterminal Gal(α1-4)Gal. Astrocytomas and Burkitt's lymphomas are thepreferred tumors as they naturally express glycosphingolipid receptors.However, any tumor expressing the appropriate receptor is appropriate.Tumor cells which express either engineered or natural functionalderivatives, or mutants of these glycosphingolipid receptors, are alsouseful. Receptor expression on the target cells is optionallyupregulated by cytokines such as IFN-γ and TNFα Tumor cell sensitivityto the cytotoxic effects of a VT is enhanced by administration ofinterleukin-1b before the addition of the conjugates. Tumor cells whichdo not naturally display Gb3 or Gb2 acquire these structures by transferfrom free, soluble structures or liposomes which express the missingglycosphingolipid receptor (Section 38, Example 5). The reconstitutedtumor cells bearing the appropriate glycolipid receptors are thustargeted for apoptosis by the above constructs and conjugates.

[0124] SAg Nucleic Acid-Verotoxin Conjugate

[0125] A preferred construct is the SAg-VT conjugate wherein the SAg ispreferably in nucleic acid form (prepared according to Example 3). TheVT portion of the complex binds to the tumor cell and initiatesapoptosis. The VT also acts as a “vector” for transfer of the SAgnucleic acid into the cell. SAg-VT conjugates bind to the terminalGal(α1-4)Gal receptors on tumor cell surfaces and are internalized viaendocytosis. The SAg nucleic acid is internalized together with the VT.The VT A chain is an RNA N-glycosidase acting on the 60S ribosomalsubunit. It induces apoptosis in the tumor cell by removing an adeninebase on amino acyl-transfer RNA so that peptide chain elongation isblocked. The resulting apoptotic tumor cells contain the internalizedSAg nucleic acid and are then ingested by dendritic cells. The DCs arecross primed to induce an effective anti-tumor response by presentingthe tumor associated antigens in the class I pathway to T cells whilethe SAg nucleic acid expresses SAg polypeptide. These activated DCs orDC/tc hybrids can be prepared by the methods of Examples 28-29. They areused to activate a T or NKT cell population in vivo as a preventativevaccine or by direct administration against established tumor. They arealso used ex vivo to produce a population of tumor specific effectorcells (T cells or NKT cells) for adoptive therapy of cancer (Examples 5,7, 14, 15, 16, 18-23, 28-29).

[0126] Glycosylation or lipid binding of the enterotoxin does notinterfere with T cell binding and activating properties. The SAg isglycosylated by chemical or recombinant techniques described in theExamples 4. The SAg glycoprotein is the further conjugated togangliosides in the ganglioside synthetic pathway via the presence ofkey signal peptides on the glyco-SAg (Example 4).

[0127] The SAg is also rerouted to the LAMP pathway, glycosylated in theGolgi apparatus and the endoplasmic reticulum and then translocated tothe membrane class II receptor as a glycosylated ganglioside.Gangliosides are glycosylated to form glycosylceramides by recombinanttechniques as described in the Example 4. They are also glycosylated byglycosyltransferases to form homologues which bind to hydrophobicregions of the SAg peptide. The final products namelySAg-glycosylceramides or SAg-LPS's then bind to CD1 receptors and areused to activate T cells or NKT cells. These construct are administereddirectly vivo or they are useful ex vivo to produce a population oftumor specific effector T cells or NKT cells for adoptive immunotherapyof cancer by protocols given in Examples 7, 15, 16, 18-23).

[0128] The present invention contemplates the fusion or coexpressionwithin the same cell of SAg polypeptides with anomeric mono anddigalactosylceramides which are expressed within a tumor cell or on thetumor cell surface. These construct could also be effectively expressedon the surface of accessory cells defined in Oxford Dictionary ofBiochemistry and Molecular Biology 1997 edition as any one of varioustypes of cell which assist in the immune response cell and includes butis not limited to DCs, fibroblasts, synoviocytes, astrocytes antigenpresenting cells, neutrophils, macrophages, basophils, eosinophils, mastcells, keratinocytes and platelets, as well as fusion cells comprisingaccessory cells and tumor cells.

[0129] The anomeric mono and digalactosylceramides have been shown toactivate NKT cells and to produce an anti-tumor response in the contextof IL-12. The galactosyl ceramides have several structural requirementsin order to produce anti-tumor effects. 12. Mono anddigalactosylceramides require an anomeric galactose or glucose as theterminal sugar or inner sugar as for example anomeric1,6-digalactosylceramide, -anomeric 1,2-digalactosylceramide, anomeric1,4-digalactosylceramide, a diglycosylceramide wherein the inner sugaris an anomeric galactose or an anomeric glucose and anomeric galactosylor anomeric glucosyl ceramide. In addition, the 3- and 4-hydoxyl groupson the phytosphingosine portion of the ceramides are preferablyunsubstituted, the sphingosine base length is preferably from about 10to about 13 carbon units and the fatty acyl chain length is preferablyin the range of about 12 to about 24 for optimal anti-tumoreffectiveness of the molecule.

[0130] The expression of anomeric mono- and digalactosylceramides in acell is achieved by several methods. The first involves the transfectionand amplification of nucleic acid encoding the enzymes which synthesizethe anomeric 1,4-, the anomeric 1,6- or the anomeric 1,2.-mono- anddigalactosylceramides such that these glycolipids are overproduced. Thegenes for these transferase enzymes have been cloned. Transfection ofnucleic acid encoding these terminal transferases into the above cellsis carried out in vivo by the methods described in Example 1.

[0131] A second method for creating cells that overexpress the foregoingglycolipids uses monensin or brefeldin which block additionalglycosylation and sialylation of the -galactosylceramides, so that themono- and digalactosylceramides accumulate in the cell.

[0132] A third approach employs cells from patients with Fabry'sdisease. These cells are genetically deficient in the -galactosidase sothey naturally accumulate -galactosylceramides.

[0133] In a forth technique, an -galactosidase deficiency is induced inthe target cell so that—galactosylceramides accumulate.

[0134] In a fifth approach, the -galactosyltransferase is transfectedFabry's disease cells, thereby adding to the usual accumulation due tothe catabolic enzyme deficiency. Such cells should have massiveaccumulations of -galactosylceramides.

[0135] In a sixth approach, the desired mono- or diglycosylceramideexpressed on liposome surfaces are transferred to tumor cells lackingthese structures by co-culture and employment of fusion techniques givenin example 5.

[0136] Nucleic acids encoding SAgs are transfected into the above cellswhich are overexpressing, overproducing or otherwise accumulating monoand digalactosylceramides. The Golgi apparatus (or Golgi complex) is amajor site of synthesis of the foregoing glycolipids. In the presentcontext, the SAg combines with it the mono and digalactosylceramides.From the Golgi, the SAg-galactosylceramide conjugates or complexes, withthe appropriate sorting signals, are dispatched in transport vesicles toother destinations. For a SAg peptide to combine effectively with an-galactosylceramide, the peptide must first have the appropriate sortingsignal which directs it to the Golgi, and from there, after complexingwith the glycolipid, to the cell surface. The trafficking pathway of SAgpolypeptide from the ER to the Golgi does not require special signals.SAg polypeptides that enter the ER (and fold and assembles properly)will automatically be transported through the Golgi apparatus to thecell surface unless they carry signals that either detain them in anearlier compartment en route or divert them (via the Golgi apparatus) tolysosomes or secretory vesicles. The SAg-glucosylceramide conjugates arerouted from the Golgi to the cell surface after acquiring a structurelike a cytoplasmic tail such as phosphoinositol which assures that thesemolecules will be bound in the cell membrane. The conjugates may also berouted to CD1 or MHC class I receptors, or via, the class II pathway, toMHC class II receptors by associating with invariant chain or LAMP-1signals as described in Section 8.

[0137] The mono- and digalactosylceramides are capable of stimulatingNKT cells (via an invariant chain) in the presence of IL-12 to producean anti-tumor response. SAgs are capable of stimulating a Tcell-dependent anti-tumor response.

[0138] The present invention utilizes tumor cells, accessory cells orhybrid cells such as DC/tc, engineered to express SAg-galactosylceramidefor anti-tumor therapy. These cells may be administered as apreventative or therapeutic vaccine (Example 29). Alternatively, theymay be useful ex vivo to activate an NKT or T cell population for use inadoptive immunotherapy of cancer (Example 29).

8. SAg Targeting to Lysosomes

[0139] LAMP-1 is a transmembrane protein localized predominantly tolysosomes and late endosomes. The cytoplasmic domain of LAMP-1 containsthe amino acid sequence Tyr-Gln-Thr-Ile whose structure conforms to theTyr-Xaa-Xaa hydrophobic amino acid motif that mediates cell membraneinternalization and possibly lysosomal targeting of several surfacereceptors. The intracellular targeting of LAMP-1 is controlled by theTyr-Gln-Thr-Ile motif located at the C terminus of its cytoplasmic tail.

[0140] In the present invention, nucleic acid encoding a SAg is fusedwith nucleic acids encoding the transmembrane and cytoplasmic tail ofLAMP-1. Nucleic acids: encoding the signal peptide (N terminal) ofLAMP-1 are integrated into this chimeric construct. These, chimericSAg/LAMP-1 polypeptides are targeted to endosomal and lysosomalcompartments, thereby rerouting transfected SAg polypeptides into theMHC class II processing pathway. Thus, cells such as tumor cellstransfected with nucleic acid encoding this modified SAg preferentiallytarget the SAg to lysosomal compartments and are presented to T cells inthe context of MHC class II. MHC class II negative tumor cells are alsotransfected with nucleic acid encoding MHC class II molecules. Theassociation of SAgs with MHC class II molecules, their natural ligandson APCs, produce optimal T cell activation to the tumor. Antigenpresenting cells transfected with these constructs are capable ofinducing potent activation of T cells. Tumor cells, in particular,transfected with this construct are administered directly in vivo orused ex vivo to sensitize a T cell population which is useful inadoptive immunotherapy of cancer by protocols described in Example 16,18-23).

10. SAg Receptors

[0141] It is clear that certain tissues express receptors forenterotoxins that are not MHC class II and that binding is reserved forselected enterotoxins and not others. Non MHC cell II binding has beenreported for colon carcinoma, mast cells epithelial cells and B cells.In a tumor bearing patient, it is desirable for administered SAgs totarget tumor cells in vivo. which naturally express enterotoxin bindingsites or receptors. Natural ligands for these receptors are nativeenterotoxins. However, because of the existence of naturally occurringenterotoxin specific antibodies in the circulation, native enterotoxinsare incapable of binding target tumor cell or T cells. The isolatedreceptor is used to screen and identify SAg proteins and/or nucleicacids which bind to the native or chimeric receptor. SAg constructs areproduced which target the tumor via its SAg receptor while alsoretaining T cell activating properties. In addition, T cells or NKTcells from tumor bearing patients are anergized in the course of tumorgrowth and are incapable of being used as a source of T cells for exvivo stimulation and adoptive immunotherapy. After transfecting thesecells with nucleic acids encoding enterotoxin receptors, they arecapable of responding to exogenous enterotoxins and are once again asource of T cells useful in adoptive immunotherapy of cancer byprotocols given in Examples 8, 9, 12, 16, 18-23.

[0142] Methods for receptor isolation purification and retrieval of cDNAare given in Example 12. The nucleic acids encoding SAg receptors aretransfected into cells by methods given in Example 1 Tumor cells have anatural binding site for exogenously administered SAg polypeptides. Inaddition, nucleic acid encoding the SAg receptors are transfected into Tcells, NKT cells, or γδ T cells of cancer patients which have beenanergized in the course of tumor growth. The expression of the SAgreceptor permits these cells to proliferate and produce TH1 cytokines inresponse to exogenous native SAg, Hence, these autologous T cellpopulations are useful in adoptive immunotherapy. Likewise, accessorycells are transfected with SAg receptor genes and used ex vivo topresent SAg to T cells Further, the nucleic acid encoding the SAgreceptor is transfected into T cells and fused, in frame, to the nucleicacid encoding the TCR-associated zeta chain or the IL-2 g to produce achimeric receptor capable of generating a signal for cell proliferationand the release of TH1 cytokines after binding its natural ligandexogenous SAg.

[0143] In one embodiment, the enterotoxin receptor is immobilized as inExample 12 and used to screen oligonucleotide libraries for binding(Gold L, J. Biol. Chem. 270:13581-13584 (1995)). Avidly bindingoligonucleotides are used to mimic the native enterotoxin by targetingthe receptor in vivo. They are coupled to the TCR binding site of anenterotoxin peptide. In this way, the hybrid molecule is administered tothe patient in a form protected from circulating enterotoxin-specificantibodies. Additionally, a nucleic acid molecule is prepared whichmimics the enterotoxin in its ability to bind to the enterotoxinreceptor on tumor cells and to the TCR on T cells. This nucleic acidmimicking the native enterotoxin is administered to the tumor bearingpatients and is capable of targeting the enterotoxin receptor sites ontumor cell and the TCR without being eliminated by circulatingenterotoxin specific antibodies as in Example 13, 18, 20-23.

11. Tumor Cells that Express SAgs and the αGal Epitope

[0144] Tumor cells are for the large part weakly antigenic and poorlyrecognized by the immune system. various attempts to increase theimmunogenicity of tumor cells by transfection of various cytokines orhistocompatibility antigens have for the most part been unsuccessful.Hyperacute rejection of xenografted organs is a very rapid and dramaticimmune event often occurring within minutes of vascularization of thexenografted organs. Very recently, a major antigenic system onxenografts which is the target of this reaction has been identified asαGalβ1-3Galb β1-4GlcNAc or α-gal. This epitope is expressed in thetissues of pigs, guinea pigs, rodents, dogs, and cows but has not beendetected in human tissue. The present invention improves theantigenicity of tumor cells and their recognition by the immune systemby providing the α-gal epitope on the cell surface either alone ortogether with SAg expression.

[0145] The αGal epitope is expressed by endothelial cells in xenograftssuch as pig organs is a major antigenic target causing hyperacute organrejection in human transplant patients. This hyperacute rejectionappears to involve a complement dependent mechanism that occurs within afew minutes. An α1-3-galactosyltransferase, is an enzyme capable ofproducing α1-3-galactose-b1-4-N-acetylglucosamine moiety by adding aterminal galactose residue to a subterminal galactose residue via anα1-3 linkage. In addition, the a1-3-galactosyltransferase is notexpressed by human and certain primate cells. Humans containxenoreactive natural antibodies that recognize αGal. For example,anti-Gal antibodies bind to pig endothelial cells that express the Galepitope. These anti-Gal antibodies are naturally occurring IgMantibodies recently found to be present in large amounts in human serum.Surface expression of the αGal epitope on tumor cells is achieved bytransfecting a cell with a cDNA clone encoding theα1-3-galactosyltransferase. While tumor cells are the preferred cellsfor transfection, other cells such as accessory cells or immunocytes arealso contemplated as being within the scope of this invention.

[0146] Nucleic acids encoding (α1-3-galactosyltransferase polypeptidesare known (Sandrin, MS et al., Proc. Natl. Acad. Sci. USA 90:11391-11395 (1993)). A cDNA clone encoding murineα1-3-galactosyltransferase is prepared using the known sequence of thisprotein and the polymerase chain reaction (PCR) technique (Dabrowski, PL et al., Transplant. Proc. 26: 1335-1337 (1994). Briefly, twooligonucleotide primers are synthesized:5′-GAATTCAAGCTTATGATCACTATGCTTCAAG-3′, which is a sense primer thatencodes the first 6 amino acids of the mature α1-3-galactosyltransferaseand contains an HindIII restriction site; and5′-GAATTCCTGCAGTCAGACATTATTCTAAC-3′, which is an anti-sense primer thatencodes the last 5 amino acids of the prematureα1-3-galactosyltransferase and contains an in-frame termination codonand PstI restriction site. These primers amplify a 1185 bp fragment froma C57BL/6 spleen cell cDNA library that is subsequently purified,digested with HindIII and PstI (Pharmacia LKB) restrictionendonucleases, and directionally cloned into HindIII/Pst I-digestedexpression vector such as CDM8 vector. After verifying the correctsequence, the α1-3-galactosyltransferase-containing expression vector istransfected into heterologous cells such as COS cells to confirmactivity. Activity can be confirmed by testing transfected cells forαGal expression using the IB4 lectin (Sigma) of Griffonia simplicifoliathat binds to Gal residues.

[0147] In the preferred mode, cells transfected with nucleic acidsencoding a SAg are co-transfected with nucleic acids that encode anα-galactosyltransferase. Alternatively, nucleic acids encoding thetransferase are transfected into a separate cell population which iscoadministered with the SAg transfected cell population.

[0148] The SAg-encoding nucleic acid can be transfected into cells whichalready express α-gal epitope. In addition, any cell can be transfectedwith the -galactosyltransferase-encoding nucleic acid. For example,α-gal-negative human tumor cells or tumor cell lines such as melanoma oradenocarcinoma are transfected with nucleic acid encoding theα-galactosyltransferase. Tumor cells transfected with-galactosyltransferase-encoding nucleic acid express the α-gal on theirsurface and are rapidly rejected when administered to a host withpreexisting α-gal specific antibodies. Methods of transfection are givenin Example 1.

[0149] Human tumor cells expressing the α-gal epitope aftertransfection, become strongly reactive with human serum containingpreexisting antibodies to the αGal epitope. Thus, an αGal-expressingtumor cell is rejected after implantation.

[0150] The ability of α-gal-transfected tumor cells to induce rejectionis demonstrated by implantation into severely compromised immunedeficient (SCID) mice that have been reconstituted with human T and Bcells and transfused with normal human plasma containing the naturallyoccurring human antibodies specific for the α-gal epitope. In this case,tumor cells transfected with -galactosyltransferase-encoding nucleicacid is rejected while untransfected cells are not. Similarly, tumorcells transfected with α-galactosyltransferase-encoding nucleic acid isrejected when implanted into species such as humans which synthesizeantibodies to the α-gal epitope compared to untransfected control tumorcells that are unaffected by the treatment.

[0151] For example, pretreatment with 10⁵-10⁷ α-galactosyltransferasetransfected tumor cells subcutaneously followed by implantation ofuntransfected tumor cells prevents the outgrowth of untransfectedmalignant tumor cells. Hence, the -galactosyltransferase transfectedtumor cells function as a vaccine. Further, -galactosyltransferasetransfected cells implanted into animals after untransfected tumors areestablished induce rejection of an established untransfected tumor.

[0152] To test for the presence of α-gal on a cell surface, α1-3galactosyltransferase knockout mice that do not express the α-galantigen are used. The α1-3 galactosyltransferase knockout mice aredescribed elsewhere (Tearle et al., Transplantation 61:13-19 (1996) andShinkel et al., Transplantation 64:197-204 (1997)). A syngeneic tumorcell that is α-gal negative such as B16 melanoma variants is transfectedwith nucleic acids that encode a given carbohydrate modifying enzyme.These transfected cells are then implanted into the knockout mouse thatreceived plasma containing α-gal specific antibodies. Tumors do not growin animals containing α-gal specific antibodies if the α-gal epitope isexpressed.

[0153] Thus, hosts implanted with αGal positive tumor cells exhibit lessgrowth than those exhibited in hosts implanted with tumor cells that areα-gal negative.

[0154] αgal negative transgenic animals are prepared which are usefulfor testing Gal expressing tumors. To produce these animals, nucleicacids encoding α-gal fucosyltransferase are transfected into α-galpositive mice. The fucosyltransferase dominates the usage of substrateN-acetyllactosamine and precludes -galactosyltransferase from utilizingthis substrate. The transgenic mice do not express α-gal on the cellsurface. In this way, transgenic mice with the H antigen rather than theGal antigen develop. Transgenic guinea pigs producing minimal -gal arealso created in this way. These animals are used as models for testingtheir capacity to reject syngeneic αGal positive tumors. These systemsalso permit the testing of α-gal specific antibodies for anti-tumoreffects after they are passively infused into animals bearing Galpositive tumors.

[0155] Neuroblastoma and some melanoma cells overexpress severaldisialogangliosides, for example, GD2 and GD3. In the present invention,nucleic acid-encoding specific sialidases or glucosidases orneuraminidases that cleave terminal sialic acid or carbohydrate residuesare transfected into cells that then express or overexpress aganglioside with an exposed αGal epitope.

[0156] Fucosylated glycolipids such as B group antigens, Lewis bloodgroup antigens, and L-selectin ligands are converted to the αGal epitopeusing the appropriate sialidases and glycosyltransferase enzymes. Forexample, a desialylating enzyme is introduced into B group antigenexpressing cells such that the α-1,3-linked galactose is exposed and nowrecognized by αGal antibodies. Mild acid treatment to remove thebranching fucose residues on the fucosylated B antigen is used to exposethe α1,3 galactose residues. Alternatively, cells expressing the Bantigen or selectin antigen are transfected with-galactosyltransferase-encoding nucleic acid that competes successfullywith fucosyltransferases for N-acetyl-lactosamine substrate andpreferentially expresses the αGal epitope

[0157] Nucleic acid encoding other polypeptides are also used to producethe surface expression of the αGal epitope such as nucleic acid encodingglycosidases that specifically cleave carbohydrate residues to exposethe αGal epitope. Tumor cells transfected with nucleic acids encodingN-acetyl-glucosaminyl transferase show an increased tendency tometastasize and colonize new organs. These same tumor cells arecotransfected with nucleic acids encoding SAgs, Staphylococcalhyaluronidase and erythrogenic toxins as well as Streptococcal capsularpolysaccharide which enables them to secrete enzymes and toxins locallyinducing a potent inflammatory and immune response at metastatic sites.

[0158] Co-transfection of tumor cells with nucleic acid encoding SAg andnucleic acid encoding a galactosyltransferase, sialidase, and/orglycosyltransferase results in expression of SAg, GalCer, αGal, or otherglycolipids on the cell surface. These tumor cells are used to stimulateT or NKT cells ex vivo to produce a population of tumor specificeffector cells which are deployed for adoptive immunotherapy of cancer.

[0159] Mutation of the glycosyltransferase nucleic acid in tumor cellsproduces a specific LPS containing the Gal/Cer or the Gal whichcoordinated with genes for protein glycosylation produce the desiredintegrated SAg LPS.

13. Tumor Cells Expressing SAgs, Glycosylceramides and LPS's and theirReceptors

[0160] It appears that anti-tumor responses are produced by asubpopulation of T cells known as NKT cells. These cells recognizeglycosylceramides with certain specifications which are presented in thecontext of CD1 receptors on antigen presenting cells. They produce IL-12mediated anti-tumor responses. Peptides of certain length withhydrophobic sequences have been shown to react with various hydrophobicregions of the CD1 receptor and produce an immune response. However,these peptides have not been implicated in an anti-tumor response. Inthe present invention, lipoproteins are contemplated which consist ofSAg or their major bioreactive domains fused to glycosylceramides in thecontext of the CD1 receptor.

[0161] To make this construct, CD1 positive cells are transfected withnucleic acids encoding glycosyltransferases that result in GalCer orGlcCer expression on the cell surface and preferably in the context ofthe CD1 receptor. The appropriate glycosyltransferase nucleic acid isobtained from Sphingomonas paucimobilis or Agelas mauritianus which areknown to express the GalCer on their cell surface. The GalCer and GlcCermoieties are recognized by NKT cell Vα invariant chains in the contextof CD1 receptors on antigen presenting cells. CD1 positive cells arecotransfected with nucleic acids encoding SAgs The resulting CD1positive cells coexpress both GalCer and SAg on the cell surface or inthe context of CD1. The GalCer and SAg presented simultaneously as acomplex and/or separate from each other on the cell surface, in thecontext of CD1 produces potent activation of NKT cells due torecognition of SAg by NKT cell Vβ chain and GalCer by the Vα invariantchain. Such GalCer-SAg complexes are loaded onto the CD1 receptor andpresented to NKT cells in this fashion. A SAg peptide capable of bindingto the TCR and activating the T cell is useful for coupling to theGalCer before or after it is positioned on the CD1 receptor. (SeeExamples 1-4, 5).

[0162] CD1 positive antigen presenting cells or tumor cells bearing theSAg glycosylceramide are used to stimulate a population of γ/δ T cellsand/or NKT cells ex vivo which is then useful in adoptive immunotherapyof cancer by protocols given in Examples 7, 15, 16, 18-23, 68. They arealso useful when, administered directly in vivo to tumor bearingpatients to produce an anti-tumor response. (See Examples 18-23).

[0163] In the present invention, nucleic acids encoding the CD1 receptorare transfected into tumor cells in vivo or ex vivo. Martin L H. et al.Proc. Natl. Acad. Sci. USA 83: 9154-9158 (1986). Nucleic acids encodingthe CD1 receptor are also cotransfected into tumor cells with nucleicacids encoding the SAg receptor. A tumor cell expressing a chimericreceptor comprising sequences of CD1 and SAg receptors is also producedby transfection of fusion nucleic acids encoding both receptors. Thetransfected tumor cell expresses either dual or chimeric receptors whichbind SAg and GalCer independently or as fusion protein or conjugate.Likewise, tumor cells are transfected with nucleic acids encoding CD14,the LPS receptor, (Ferrero, E. et al., J. Immunol. 145: 331-336 (1990))a leucine rich receptor glycoprotein found on myeloid cells with a LPSbinding site between amino acids 57-64. Nucleic acids encoding CD14 aretransfected into tumor cells together with nucleic acids encoding SAgsand resulting tumor cell expresses several receptors or a singlechimeric receptor with preserved consensus binding sequences common toeach. These tumor cell transfectants are capable of binding exogenousSAg and/or LPS and or GalCer. The resulting tumor cells with bound SAg,and/or Gal/Cer and/or LPS activate a population of T cells, γ/δ T cellsand/or NKT cell to produce tumor specific effector cell which are usefulin the adoptive immunotherapy of cancer by methods in Example 1-7, 12,15, 16, 18-23, 68). The tumor cell transfectants are also administeredas a vaccine or to hosts with established tumors as in Example 19-23.

[0164] Alternative splicing and utilization of cryptic splice sitesgenerates alternative reading frames and secretory isoforms of CD1, CD14and SAg receptors. Woolfson A. et al., Proc. Natl. Acad. Sci. USA 91:6683-6687 (1994). These soluble receptors are immobilized on solidsurfaces such as polystyrene plates or beads and bind their respectiveligands e.g. GalCer and SAg. In this form, the GalCer and SAgs activateT cell or NKT cell to produce a population of tumor specific effector Tcell, γ/δ T cell, NK cell or NKT cells useful in adoptive immunotherapyof cancer by methods given in Examples 7, 15, 16, 18-23, 65).

14. SAg-Activated Tumor Specific T Cells, NKT Cells or γ/δ T CellsExpressing CD44 for Adoptive Immunotherapy

[0165] It is imperative that T cells, NKT which are stimulated in vivoor ex vivo by the SAg constructs after SAg stimulation, is an indicatorof upregulated adhesive capacity which is requisite for the homing ofSAgs to tumor sites. T cells or NKT cells or cells transfected withnucleic acids encoding SAg receptors i.e., tumor cells or accessorycells are stimulated by SAgs in vivo or ex vivo to express CD44. TheseCD44 expressing T cells are enriched and expanded and then harvested foruse in adoptive therapy of cancer by protocols given in Examples 7, 15,16, 18-23.

[0166] Transfection of cDNAs encoding soluble isoforms of CD44 intotumor cells results in the local release of soluble CD44 which inhibitsthe ability of endogenous cell surface CD44 to bind and internalizehyaluronate and to mediate tumor cell invasion. Mice injected with tumorcell transfected with the CD44 isoform showed not tumor metastases. Suchtumor cells were shown to undergo apoptosis. These transfectantsdisplayed a marked reduction in their ability to internalize and degradehyaluronate. Therefore, CD44 function promotes tumor cell survival ininvaded tissues possible as a result of impairing their ability topenetrate the host tissue hyaluronan barrier. In the present invention,SAg-encoding nucleic acid is co-transfected or fused to nucleic acidsencoding CD44 isoforms. These transfected cells are capable of migratingto sites of metastatic tumor in tumor bearing hosts and eliciting apotent anti-tumor response. The combined apoptotic effect to the CD44isoform with the enhanced immunogenicity of the SAg produces a powerfulsynergistic anti-tumor response. The nucleic acids encoding the CD44isoform and SAg are transfected into accessory (DC)/tumor cell hybrids.In addition, to presenting tumor antigen and SAg to the immune systemand inhibiting metastases, the CD44 isoform produces apoptosis of thefusion cell which in turn is ingested by DCs resulting in enhancedimmunogenicity and a more potent tumoricidal response. These combinedtransfectants are used preferably against established tumor according toprotocols in Example 19-23.

[0167] NKT cells or T cells that do not produce CD44 after SAgstimulation do so after transfection with nucleic acids encoding CD44 ortransferases such as N-acetylglucosaminyl transferase III or CD44(Sheng, Y. et al., Int. J. Cancer 73, 850-858 (1997); Nottenberg, C. etal., Proc. Natl. Acad. Sci. USA 86: 8521-8525 (1992)). The latter enzymesynthesizes bisecting N-acetylglucosamine structures on asparaginelinked oligosaccharides. Glycosylation of CD44 by these transferasesproduces enhanced CD44 mediated adhesion to immobilized hyaluronate.SAgs are used to activate T cells which have been transfected withnucleic acids encoding N-acetylglucosaminyltransferase III. The SAgstimulated transfectants display increased CD44-mediated adhesion. aswell as lymphocyte homing and trafficking. Certain T cell, NKT cell or/Tcell populations which are unable to express CD44 after SAg stimulationare transfected with nucleic acids encoding CD44 before sensitizationwith SAgs. These cells express CD44 after immunization with SAgs in vivoor in vitro. These additional populations of effector T cells are usefulin adoptive immunotherapy of cancer by methods given in Examples 5, 7,15, 16, 18-23.

15. Tumor Associated Antigens

[0168] Tumor associated antigens include:

[0169] (1) Normal structures, e.g., differentiation or tissue specificantigens,

[0170] (2) Mutated normal structures

[0171] (3) Products of alternate reading frame or fusion of severalgenes

[0172] (4) Chimeric products resulting from cell or gene fusion

[0173] (5) Xenogeneic antigens (“xenoantigens”)

[0174] A tumor antigen (also called “tumor associated antigen) is anyantigenic structure expressed by a tumor cell. For example, tumorantigens include mutated products of various oncogenes and p53 genesthat are expressed in tumor cells generally. Many tumor antigensassociated with particular types of cancers are known. For example,tumor antigens associated with breast, colon, and lung cancer are knownand have been cloned. Common melanoma antigens recognized by Tlymphocytes have been identified and are used as immunotherapeuticantigens for treatment of melanoma. Five genes encoding differentmelanoma antigens have been identified. For example, MAGE1 and 3,expressed on melanoma and other tumor cells, are recognized by cytotoxicT lymphocytes (CTL) in the context of HLA-A1 (Van der Bruggen P et al.,Science 254:1643 (1991) and Gauler B et al., J. Exp. Med. 179:921(1994)). MART-1 identical to Melan-A (Kawakami et al., Proc. Natl. Acad.Sci. USA 91:3515 (1994) and Coulie et al., J. Exp. Med. 180:35 (1994));gp100 (Kawakami et al., Proc. Natl. Acad. Sci. USA 91:6458 (1994)); andtyrosinase (Brichard et al., J. Exp. Med. 178:48 (1993)) are melanocytelineage-related antigens expressed on both melanoma and melanocytes.MART-1 and gp100 have been shown to be recognized by MHC-classI-restricted CTL in the context of HLA-A2, and tyrosinase in the contextof HLA-A2 and HLA-A24 [Robbins et al., Cancer Res. 54:3126 (1995)]. Anadditional list of tumor antigens useful in this invention is given inRosenberg, SA. “Principles and Applications of Biologic Therapy inCancer,”: In: Principles and Practice of Oncology, DeVita, VT et al.,eds, J. B. Lippincott Co., Philadelphia, Pa. 1993.

[0175] In addition tumor associated antigens are defined as includingnormal structures expressed in tumor cells, mutated normal structures,normal differentiation- or tissue-specific structures, products ofalternate reading frames of the same genetic regions, chimeric productsof several genes that originated in a parental or in a fused, hybridcell. This also includes gene products expressed in association with MHCmolecules or other surface receptors, organelles or vesicles.

[0176] Tumor cells expressing tumor-associated antigens are transfectedin vivo or ex vivo with nucleic acids encoding a SAg alone or togetherwith nucleic acids encoding other products, such as those listed inTables I and II. These include surface antigens and receptors such asthe Gal epitope, GalCer, CD1, CD14 and SAg receptor.

[0177] The transfected cells may be of host origin, or syngeneic,allogeneic or xenogeneic; the cells may be non-malignant. SAg-encodingnucleic acid may also be inserted into a mutated normal gene in a tumorcell, e.g., LDL receptor gene in a melanoma cell. The LDL receptor isexpressed as a fusion product of the LDL receptor gene (chromosome 19)and a fructose transferase gene on the same chromosome. This combinationresults from chromosome inversion which gives rise to the fusion productprobably due to recombination between the two ends of this chromosome.The expressed peptide epitope is therefore a nonsense sequence beingread in the wrong direction. The three base pair mutations in the thirdopen reading frame results in the expression of a mutant peptide. Sitedirected mutagenesis can be achieved by insertion of SAg-encodingnucleic acid into the mutant gene at any feasible site or by targetinginsertion in place of the mutated base pairs. The resultant LDL receptordisplays the SAg alone or as a chimera with the mutant sequence. Sitedirected mutagenesis by SAg-encoding nucleic acid may also target theβ-catenin gene which in melanoma shows a single C-T mutation whichresults in a ser to phe substitution and the generation of the 9 aminoacid mutant peptide. The SAg-encoding acid may be inserted or maysubstitute for any sequence in a normal non-mutated gene, atissue-specific or differentiation-associated gene in tumor cells, orother genes expressing their products in a tumor cell. Preferably, themutated gene product is immunogenic and recognized as a dominant epitopeby the host immune system, preferably by T cells (including tumorinfiltrating lymphocytes). The mutated sequence may, in contrast, be aweak immunogen which is rendered more immunogenic when presented in thecontext of a SAg.

[0178] In the preferred embodiment, the transfected cells are tumorcells of host origin expressing a defined tumor associated antigen suchas MART-1. If the tumor antigen is not expressed or weakly expressed onthe transfected cells, then the tumor cell is transfected with nucleicacids encoding an immunogenic tumor antigen such as MART-1, tyrosinaseor MAGE-1 in addition to SAg and other constructs described herein.

[0179] The tumor cells may be transfected in vivo by administeringnucleic acids encoding SAgs and/or the other nucleic acid constructsdescribed above using a site directed mutagenesis approach in vivo andmethods such as described in Example 1, 3, 18-23. Tumor cells may alsobe transfected ex vivo by methods given in Example 1-3. Ex vivotransfected tumor cells are used as vaccine or to treat establishedtumor by methods and protocols in Example 18-23 They are also useful exvivo to immunize T cells, γ/δ T cells, NK cells or NKT cells to producea population of tumor specific effector cells adoptive immunotherapy ofcancer by methods and protocols given in Examples 7, 15, 16, 18-23, 68.

16. Immunostimulatory Sequences

[0180] Several of constructs consist of nucleic acids encoding SAgpeptides which produce anti-tumor responses by activating host TH-1 CD4+T cells to proliferate and produce tumoricidal cytokines such as IL-1α,IL-1β, IL-2, IL-6, TNF-α, TNF-β and IFN-γ. The incorporation of theimmunostimulatory sequence into the genetic construct of SAg DNA,ensures that the T cell response is skewed to produces a predominantproliferation of TH1 cells and production of a TH1 cytokine profile.Immunostimulatory sequences (ISS) consist of DNA sequences that exhibitimmunogenicity. Briefly, plasmid DNA (pDNA) having shortimmunostimulatory DNA sequences containing a CpG dinucleotide in aparticular base context were shown to be immunogenic (Tokunaga J et al.,J. Natl. Cancer Inst. 72:955-962 (1984)). By synthesizing singlestranded nucleotides corresponding to different regions in theMycobacterium bovis genome, specific single stranded oligonucleotidesthat activate adherent splenocytes and enhanced natural killer cellactivity have been identified. In addition, single strandedoligonucleotides with CpG motifs induce B cell proliferation andsecretion of IL-6 and IFN (Krieg et al., Nature, 374:546 (1995)). Theactivation capability generally has the formula5′-Pur-Pur-C-G-Pyr-Pyr-3′. Further, human monocytes transfected withpDNA or double stranded oligonucleotides containing ISS transcribedlarge amounts of IFN-γ and IL-12 (Sato et al., Science 273:352-354(1996); Zhu et al., Science 261, 209-211, (1993)) Direct gene transferwith plasmid-cationic liposome complexes resulted in lasting,generalized or tissue specific expression of the injected geneticphenotype. Similarly, any RNA or DNA vectors carrying nucleic acidsencoding SAg alone or associated with additional therapeutic nucleicacids which are enhanced by optimizing promoters, introns, andpolyadenylation signals (Donnelly, J J et al. Annu. Rev. Immunol. 15:617-648 (1997)) are also useful in augmenting expression of SAg andenhancing an anti-tumor effect.

[0181] In the present invention, the ISS is inserted into nucleic acidsequences of SAgs and tumor associated antigens which are used totransfect tumor cells, antigen presenting cells, accessory cellsincluding muscle cells in vitro or in vivo by methods given in Example1-3, 15, 16, 18-23. In all instances, the SAg stimulation of the T cellresponse is critical to an effective anti-tumor response of the host.The presence of the ISS ensures that the SAg nucleic acidspreferentially activate the TH1 after in vivo administration of thenucleic acids encoding SAg. SAg DNA is useful ex vivo in activating Tcells, γ/δT cells and NK cells and NKT cells by direct transfection orby presentation via incubation with pretransfected antigen-presentingcells or tumor cells. The tumor specific T effector cell are then usefulfor adoptive therapy of cancer using protocols given in Examples 7, 15,16, 18-23, 68). A particularly useful method involves the intratumoralinjection of nucleic acids encoding SAgs. The latter is administered innaked, plasmid or liposomal form. Once tumor inflammation is initiated(generally within 15 days after injection), the host is given T cells orNKT cells which have been immunized in vitro to the tumor by tumor cellstransfected with nucleic acids encoding SAg plus additional constructsgiven in Tables 1 and II by methods given in Examples 7, 15 16 18-23.

17. Liposomes

[0182] Liposomes containing repeating units of the α-gal epitope,GalCer, and/or SAgs are constructed and administered directly into atumor. These elements are combined before incorporation into liposomesor they are added individually in the preparative procedure. Methods forpreparation of these liposomes are given in Examples 5. These liposomesare preferentially delivered parenterally or directly into the tumor.The administration of SAgs in this manner provides a high localconcentration of SAg to stimulate an anti-tumor response. Theseliposomes are also useful ex vivo by activating a T cell, γ/βT cell, NKor NK T cell population which is then harvested and used for adoptiveimmunotherapy as described in protocols in Examples 5, 7, 15-17, 18-23,68).

18. Tumor Cells that Induce Cellulitis

[0183] Transfection with microbial nucleic acids that encode tissuespreading factor (hyaluronidase), erythrogenic toxins, enterotoxins;capsular polysaccharides from S. aureus and Streptococcus pyogenes, S.aureus and S. pyogenes have potent tissue invasive properties.Specifically, Staphylococcus and Streptococcus are capable of invadingtissues by secreting several enzymes which lyse ground substance such asmucopolysaccharide, hyaluronic acid, or chondroitin sulfate, createlocal thrombosis, and initiate inflammation and edema. These enzymesconsist of hyaluronidase, streptokinase, streptodornase, erythrogenictoxins as well as various enterotoxins (Example 3). In the presentinvention, the nucleic acid sequences encoding these potent enzymes aretransfected into tumor cells, either in vitro or in vivo (Examples 1-3,6, 15, 16, 18-23). In vivo, the transfected tumor cells migrate to sitesof existing metastases. The transfected tumor cells secrete the enzymeswhich hydrolyze the tumor ground substance and neovasculature and toxinsto induce inflammation and an immune response in tumor tissue. Tumorswhich are encased in nests of connective tissue are eliminated by thisprocess. The resulting increase in local vascular permeability inducedby the combined effect of enzymes and toxins produces intenseinflammation at tumor sites. If their administration is timed to thepeak of tumor inflammation, liposomes as described herein andchemotherapy are sequestered and concentrated in the inflamed tumor bedproducing an, augmentation of the tumoricidal response.

[0184] A relatively low number of transfected tumor cells with thecomplete microbial enzymatic and toxin genetic construct would berequired to induce a tumoricidal effect. The population of transfectantswould then proceed to secrete these microbial enzymes locally. Inaddition, nucleic acid encoding these enzymes are derived from a strainof Staphylococcus or Streptococcus such as Staphylococcus epidermidis orStreptococcus bovis of low or intermediate virulence.

[0185] Tumor cells are cotransfected with glycosyltransferases ortreated with glycosyltransferase-inducing agents resulting in theexpression of the α-gal epitope and reduction in the survival time oftumor cells For example, the nucleic acids encoding theglycosyltransferase from Sphingomonas paucimobilis or Agelas mauritianusproduce GalCer are transfected into tumor cells to induce the surfaceexpression of GalCer or α-gal. The tumor cells then express and/orsecrete microbial agents such as SAgs, hyaluronidase and erythrogenictoxins that hydrolyze the ground substance of the tumor. By alsodisplaying SAgs and -Gal or Gal/Cer epitopes which activate NKT cells, Tcells, and Gal specific antibodies the transfected tumor cells induceprofound tumoricidal activity. These transfected tumor cells are used toactivate a population of T cells including αβ T cells γ/δ T cells, NKand NKT cells to become tumor specific effector cells which are employedfor the adoptive immunotherapy of cancer. See Examples 1, 2, 4-5, 7, 15,16, 18-23.

[0186] For in vivo transfection of tumor cells, the microbial geneticnucleic acids are targeted to tumor cells as described herein (See p. 12“Transfection”, Examples 1-3, 6, 19). Once localized in tumor sites invivo, the tumor cell is capable of hydrolyzing surrounding stroma and,initiating thrombosis, inflammation, and increased tissue permeability.Additional microbial nucleic acid encoding proteinases, lysoproteinases,tissue spreading factors, a and b hemolysins and toxins are alsotransfected into tumor cells and used in accordance with this invention.

[0187] Micrometastatic disease in cancer patients is of great concern asit often goes undetected and is refractory to chemotherapeutic agents.Documented metastases in breast cancer patients is associated with apoor prognosis. The present invention contemplates that the metastaticproperties of tumor cells coupled with the potent inflammatoryproperties of the microbial products are useful in tracking andeliminating micrometastatic disease in tumor bearing patients. Tumorcells are transfected with nucleic acid encoding polypeptides involvedin metastasis. These include but are not limited to, peptides thatupregulate the adhesive properties of CD44 (e.g., glycosyltransferases),the c-erbB-1 encoded EGF receptor which is associated with enhancedmetastases in breast carcinomas or c-erbB-2/neu encoding the p185receptor associated with poor prognosis in breast and ovariancarcinomas. These cells with metastatic activity are programmed totraffic, home and colonize specific sites of existing metastases thetumor bearing host. Hence they have the unique property of charting themicrometastatic sites of the tumor. These tumor cells are cotransfectedwith microbial nucleic acids encoding the hyaluronidase, erythrogenictoxin and enterotoxins as well as the α-gal. Hence, as they colonizemetastatic sites, these transfectants induce a potent inflammatory andimmune response. This ensures their own destruction together with thesurrounding untransfected micrometastatic tumor cell population andneovasculature and stroma. Methods of preparation, administration andassessment of these transfectants in tumor bearing hosts are in Example1-3, 18-23.

[0188] The tumor cells are also transfected with the above microbialgenes on a DNA template with a tissue specific promoter in order totarget the activity of these transfected tumor cells to the vital organs(and sites therein) affected by the existing metastatic tumor. Forbreast cancer, this would be lung, liver or brain. These organ-specificpromoters ensure that the expression of the microbial products wouldoccur in the organ(s) targeted by the tissue specific promoter

[0189] The same tumor cells are also provided with inducible promotersequences which control the level of receptor transcription andexpression. Inducible promoters suitable for use in mammalian cellsinclude the MMTV-LTR under the control of steroid hormones and themetallothionein promoter under the control of heavy metal ions. In thiscase, the microbial nucleic acids are linked to steroid inducible genesequences. Transcription is triggered when these cells are exposed to athreshold level of steroids. Hence, two to three days afteradministration to the host, when the above transfectants have colonizedtumor metastatic sites, a bolus of corticosteroid is administered whichinitiates transcription of the microbial enzymes and toxins by the tumorcell transfectants and their secretion. In this fashion, the transfectedtumor cells express and secrete their inflammatory products inmetastatic tumor sites resulting in the elimination of metastaticdisease.

19. Tumor Cells as Mimics of Virulent Bacteria: Transfection withNucleic Acid Encoding Bacterial Invasins, Virulence Factors, and Enzymesthat Degrade Extracellular Matrix

[0190] Tumor cells with a metastatic phenotype are transfected withnucleic acids encoding proteins with the capacity to invade and adhereto inflammatory cells such as macrophages (adhesins and virulencefactors). These genes are inducible and controlled by operons.

[0191] SAg-encoding nucleic acid is fused in frame to nucleic acidencoding oncogenes involved in tumorigenesis and metastasis. Examples ofsuch genes, in addition to erb/neu, crb, crbB2 and EGF (epidermal growthfactor receptor) discussed above, include ras and mutated ras, erk, andmtal, 182 mts1, nm23 (See p 181 of Franks L. M. et al., Cellular andMolecular Biology of Cancer, Oxford University Press, Oxford UK, (1997)which is incorporated by reference), as well as the laminin-integrin andthe cadherin family. These genes are particularly useful because theyare overexpressed in tumor cells displaying a metastatic phenotype.

[0192] Invasins SAg-encoding nucleic acid is fused in frame orcotransfected into tumor cells with nucleic acids encoding bacterialinvasins and hyaluronidases. The invasin imparts leukocyte like activityto bacteria is transfected into tumor cells which allows the tumor cellsto penetrate tissues. These are exemplified by Yersiniapseudotuberculosis invasin and hyaluronidase (including its variousisotypes) and also known as tissue spreading factors. The invasin geneexemplified in Y. pseudotuberculosis encodes a protein located in theouter membrane of the bacterium called invasin (Inv) and the gene isknown as inv. The DNA region of the inv gene contains a open readingframe 2964 bases. This protein binds to the host cell surface by meansof the C-terminal 192 residue region. Mutation by insertion of atransposon or elimination of the inv gene greatly impairs the ability ofthe bacterium to penetrate tissues (Schaecter M et al., Genetics ofBacteria edited by Baer G M et al., in Mechanisms of Microbial DiseaseWilliams and Wilkins Baltimore (1993)).

[0193] The host membrane receptors for invasin belong to the integrinsuperfamily with a particular affinity for VLA-3, 4, 5, 6. Invasin alsobind to T cell α₄β which is involved in lymphocyte homing or traffic.Once bound to a phagocyte, phagocytosis is triggered and the bacteriumis taken up. Nucleic acids encoding Inv are transfected into tumor cellswhich confers upon the tumor cell a phagocytosis triggering signal forhost macrophages.

[0194]E. coli genes of the P pili or pap operon encoding adhesinproteins have been isolated from chromosomes and plasmids. The genecluster is linked to genes for other virulence determinants such as theKI capsular polysaccharide and hemolysin. The receptor for the pili isthe αGal(1-4)Gal moiety of the P blood group antigen. Examples of hostcell receptors for bacterial adhesins is given in Table 7.2 of Patrickand Larkin. Pilin genes in N. meningitidis encode proteins is which thefimbriae are the N-methylphenylalanine pili. An extensive region ofamino acid homology at the N-terminal end is common to a wide range ofbacterial genera including Pseudomonas aeruginosa, N. gonorrhoeae, N.meningitides, Moraxella bovis and Bacteroides nodosus. This N-terminalregion is highly hydrophobic which is in contrast to the fimbriae of theEnterobacteriaceae which either have a hydrophobic region at theC-terminal end or lack a hydrophobic region altogether. Of interest isthe presence of a site on SAgs which resembles the third Ig-likedisulfide-bridged loop of VCAM-1 and a conserved sequence is presentwithin the same subregion of the fifth Ig-like VCAM-1 loop. The onlyknown receptor for the VCAM-1 is VLA-4, an adhesion molecule expressedprimarily by activated T and B cells. A survey of target cellsusceptibility to SEC dependent lysis shows a correlation between VLA-4expression and susceptibility to lysis.

[0195] Superantigens Fused to Invasins

[0196] Superantigens are fused to nucleic acids encoding invasinmolecule or the integrin binding sequences of the invasin molecule.Invasin is a 986 amino acid outer membrane protein that tightly binds atleast five different β,-chain integrins prior to uptake in-culturedcells (Isberg et al., 1987; Isberg et al., 1988, E. Krukonis et al.,unpublished observation). The C-terminal 192 amino acids of the proteincontains the integrin binding activity of invasin (Leong et al., 1990)and this region is also sufficient to promote entry when used to coatnon-invasive bacteria, such as S. aureus (Rankin et al., 1992). TheN-terminal portion of invasin is required for secretion to and properlocalization within the bacterial outer membrane (Leong et α, 1990).Integrins are a large family of heterodimeric receptor'that mediate avariety of cell-cell and cell-extracellular matrix interactions (Hynes,1992). In addition, residues within an 11-amino acid region encompassingresidues 903-913 are also critical for cell binding. One of theseresidues, aspartate 911 (Asp-911), appears to be absolutely essentialfor binding, as changes at this position result in complete loss ofintegrin binding.

[0197] The Yersinia pseudotuberculosis invasin protein mediatesbacterial entry into mammalian cells by binding multiple β,-chainintegrins. Integrins are a large family of heterodimeric receptors thatmediate a variety of cell-cell and cell-extracellular matrixinteractions (Hynes, 1992). Invasin binding to purified α₅β_(L),integrin is inhibited by Arg-Gly-Asp (RGD)-containing peptides, althoughinvasin contains no RGD sequence.

[0198] The inv gene has been cloned (Young V B et al., Mol. Microbiol.4: 1119-1128 (1990). Fifteen mutations that diminished binding andbacterial entry were isolated after mutagenesis of the entire inv gene.All of the mutations altered residues within the C-terminal 192 aminoacids of invasin, previously delineated as the integrin binding domain,and 10 of the mutations fell within an 11 residue region. When thissmall region is subjected to site-directed mutagenesis, almost half ofthe 35 mutations generated decreased invasin-mediated entry. D911 withinthis region is the most critical residue, as even a conservativeglutamate substitution abolished bacterial penetration.

[0199] The fusion protein consisting of a superantigen (SEs, SPEs orYersinia and other non SE or SPEA, superantigens) fused to an invasinare prepared as in Example 5. They are used for treatment of establishedand metastatic tumor or as a preventative vaccine as described inExamples 14-16, 18-23.

[0200] Hyaluronidases and Proteases

[0201]Bacteroides species produce hyaluronidase, heparanase, andchondroitin sulfatase enzymes. C. perfringens m toxin is a hyaluronidaseenzyme and Bacteroides and C. perfringens produce elastase andcollagenase enzyme while Porphyromonas gingivalis has a cell associatedcollagenase. Streptococcus pyogenes produces hyaluronidase enzymes whichdepolymerize their own capsules. Neuraminidases and endoglycosidases,lipases, nucleases and proteases produced by a wide variety of bacteriaare also useful in this invention as capable of promoting tissuenecrosis in tumor masses and/or tumor nests.

[0202] The staphylococcal invasive genome is predominantly chromosomaland the nucleic acid segments encoding the major invasive enzymesystems, permeability factors, and toxins have been isolated, cloned,and sequenced. For example, the nucleic acid sequence encoding ahyaluronidase from group A Streptococcus strain 10403 is describedelsewhere (Hynes et al., Infect. Immun. 63:3015-3020 (1995)). Tumorcells transfected with nucleic acids encoding microbial invasive andinflammatory substances are preferentially used in vivo where they areprogrammed to traffic to metastatic sites and/or organs primarilyinfiltrated by the tumor. Once situated in tumor, they commencesecretion of their inflammatory enzymes and toxins. Protocols for theirpreparation, use, and assessment are given in Examples 1-3, 18-23.

[0203] Consolidation of Bacterial Genes

[0204] The microbial nucleic acids encoding hyaluronidase, erythrogenictoxins proteases, coagulases and enterotoxins are consolidated into achimeric construct or plasmid and transfected into tumor cells whichthen commence secretion of the spreading factors, pro-inflammatory andpermeability inducing agents. For example, a single construct ormultiple constructs contains the nucleic acid encoding polypeptidesincluding, without limitation, enterotoxin B, hyaluronidase,streptokinase, coagulase, Staphylococcal protease and erythrogenictoxins.

[0205] Tumor cells transfected with the above microbial genes areprepared as in Example 1-3 and are used in the treatment of establishedand metastatic tumor or as a preventative vaccine as described inExamples 15-23.

20. Combined Expression of Different Stimulatory Molecules byCo-transfection of Tumor Cells or Fusion of Singly Transfected Cells

[0206] Tumor cells that express two different types of exogenousmolecules are produced by either cotransfection of the same cells with(a) SAg-encoding nucleic acid and (b) nucleic acid encoding a toxins orautolysin, or by fusion of tumor cells that have been singly transfectedwith (a) with tumor cells transfected by (b) Tumor cells are providedwhich have the dual capacity to colonize metastatic tumor sites in vivoand induce inflammation. Once situated in sites of tumor metastasis, thetumor cells behave like a necrotizing bacterium or leukocyte. Forexample, tumor cell are transfected with nucleic acids encodingbacterial invasins to promote adhesion, “tissue spreading factor” orhyaluronidase to hydrolyze the ground substance, coagulase to inducelocal thrombosis and streptokinase and streptodornase. In addition,tumor cell are provided with nucleic acids encoding bacterial toxinswhich bind and produce autolysis and cytotoxicity for surrounding tissueand tumor cells. The tumor cells are also cotransfected with additionalnucleic acids encoding SAgs. The toxin genes useful herein are amplifiedby providing two copies tandemly duplicated on a chromosome and linkedto an amplified oncogene. Situated in tumor tissue, these transfectedtumor cells release enterotoxins as well as inflammatory enzymes,immunogenic capsular lipoproteins, cell wall LPS's and cytolysins. Thisevokes a potent T cell and inflammatory response in tumor tissue. Theseinflammatory genes are inducible at the level of the operon or in someinstances bacteriophage which controls their activation. Transfectedtumor cells are transfected with microbial nucleic acids given aboveeither in vitro or in vivo at tumor sites as in Example 1-3, 5, 16-23and p.11 under “transfection”.

[0207] The S. aureus α toxin forms pores or transmembrane channels in awide range of host cells. It is released from the bacteria duringexponential growth and has a molecular mass of 33 kDa. Expression of thegene encoding the a toxin, hly, is under the control of the agr genewhich coordinately controls the expression of a number of extracellularproteins, including exfoliatin toxin, toxic shock syndrome toxin, a, b,and d toxins, enterotoxin B, lipases and nucleases. The β toxin is aphospholipase which attacks a sphingomyelin in the cell membranes. Thephage encoding the toxin is hlb. Exfoliatin toxin A is encoded by achromosomally located gene eta and the gene for toxin B is etb. The etagene is by the agr gene regulator which is a member of thehistidine-protein kinase response regulator superfamily. (Patrick S etal., Immunological and Molecular Aspects of Bacterial Virulence, JohnWiley and Sons New York, N.Y. 1995)

[0208] SEB binds to glycosphingolipids on cell membranes. Theganglioside binding site on SEB is overexpressed, or a myristoylationsite or GPI binding site is integrated into its structure so that it isbound to the surface of the tumor cell membrane and not secreted. TheSEB will preferentially bind to tumor cell expressing ganglioside tumorassociated antigens and will augment the immunogenicity of theseantigens.

[0209]S. aureus produces a bifunctional protein autolysin of110-kDa,(HlyA) via the atl gene that has an N-acetylmuramoyl-L-alanineamidase domain and an endo-β-N-acetylglucosaminidase domain. Itundergoes proteolytic processing to generate two extracellular enzymesthat are secreted. The specific secretion proteins HlyB and HlyD are 80kDa and 54 kDa respectively. The process is directed by the hlyB andhlyD genes which are contiguous and co-expressed with the hylC and hylAgenes that are required for the synthesis of protoxin and the acylcarrier protein-dependent fatty acylation that matures it tocytolytically active toxin. Hemolysin is secreted as the mature acylatedform of the hlyA gene product proHlyA following the covalent attachmentof a fatty acid moiety in a cytoplasmic mechanism directed by thedimeric HlyC activator, a putative acyl transferase and dependent uponthe acyl carrier protein. This specific and novel HlyC-directed fattyacylation is required to target the hemolysin toxin to mammalian cellmembranes prior to forming cation-selective pores and disrupting thehost cell.

[0210] Bacteria such as E. coli, Bordetella pertussis, Pasteurellahaemolytica, Proteus vulgaris and P. mirabilis produce geneticallyrelated toxins. Their activity is dependent on the presence of calciumions. Characteristically, they have regions of 10 to 47 repeats withinthe amino acid sequence and termed repeats in toxin or RTX gene family.The repeat sequence contains the following nine amino acids;leucine-X-glycine-glycine-X-glycine-asparagine-aspartic acid-X where Xis a variable amino acid. These repeats are required for hemolyticactivity. A large hydrophobic region of the hemolysin separate from therepeats, is also essential for activity and may be involved in theinteraction with the host cell membrane. The hemolysin A of E. coliapparently form pores on the target cell membrane. This requires a 20kDa product of another gene HlyC before it becomes actively hemolytic.In E. coli, the operon for the production of the hemolysin contains fourgenes hlyA which codes for the structural hemolysin and hlyC which isrequired for activation of the HlyA. The other two genes hlyB and hlyDare involved in the transport of HlyA to the extracellular environment.Pasteurella haemolytica leukotoxin and Bordetella pertussis adenylatecyclase hemolysin have similar C-terminal sequence and associated genesanalogous to those in the hly operon. (Koronakis V et al., Secretion ofHemolysin and other Proteins out of the Gram-Negative Bacterial Cell, inGhuysen J M et al., ed, Bacterial Cell Wall, Elsevier, Amsterdam(1994)).

[0211] The Shiga toxin of Shigella dysenteriae and Shiga-like toxins ofE. coli (Verotoxins) are a family of related toxins which have similaramino acid sequences and biological activities. The A subunit of Shigatoxin has a molecular mass of 31 kDa which associates with five to the 7kDa B subunits. The A subunits is proteolytically cleaved into A1 andA2. It is the A1 fragment which is biologically active. The host cellreceptor for Shiga toxin is the glycolipid Gal(α-4)Gal(β1-4) GlcCeramide(globotriosylceramide; Gb3) and for Shiga-like toxin I (SLTI) and SLTIIof E. coli is Gal(α-3)GalCeramide (Galabiosylceramide). The bindingspecificity is dependent on both sugars residues and the lipid moiety.The Shiga toxin is known to inhibit protein synthesis. It is a RNAN-glycosidase enzyme whose site of action is the 60S ribosomal subunit.The toxins remove an adenine base from position 4324 on the aminoacyl-transfer RNA binding site of 28S ribosomal RNA hence preventing peptidelength elongation. The effect on protein synthesis is similar to that ofdiphtheria toxin and Pseudomonas aeruginosa exotoxin A. The SLTI and IItoxins of E. coli and encoded by lysogenic phage. Its expression iscontrolled by iron concentration in the growth medium by way of the furgene and iron box repressor protein binding site. Clostridia difficiletoxins A and B also bind to anomeric galactose epitopes on cellmembranes and induce membrane associated enzymes and inhibit G proteinactivation which results in cell death. Tumor cells transfected with agalactosyltransferase genes to produce the a α-galepitope aresusceptible to lysis by both the Shiga-like toxins and C. difficiletoxin. The expression of the α-gal epitope is enabled by thetransfection of nucleic acids encoding -Gal transferase into tumorcells.

[0212] Listeria monocytogenes produces a hemolysin. listerolysin O(LLO), a member of the thiol-activated family of cytolysins. LLO isencoded by the gene hyl (also designated hylA and lisA). Listerolysin Otoxin is a pore forming toxin which degrades the membrane of itsphagocytic vacuole allowing the bacterium to escape into the hostcytoplasm. This gene cloned into Bacillus subtilis enables the bacteriumto grow rapidly intracellularly in the cytoplasm of a macrophage-likecell line after disrupting the phagosomal cell membrane.

[0213] Tumor cells are transfected with the above microbial nucleicacids as in Example 1-3. These transfectants are useful in vivo againstestablished tumor and micrometastatic disease (Examples 5, 15, 16,18-23).

21. Augmentation of Tumor Cell Immunogenicity by Bacterial Products:Transfection with Genes Encoding Bacterial Antigens or Receptors forBacterial Products

[0214] Tumor cell are provided with augmented antigenicity by expressingfundamental patterns that are recognized by fundamental recognitionunits of the innate immune response. Examples are LPS's of gram negativeorganisms, SAgs and peptidoglycans of gram positive organisms, fungalβ-glucans, bacterial glycosylceramides, and mycobacterial lipoarabinans.Numerous infectious agents with these structures cause potent immunereactions e.g. streptococcal cellulitis induced by S. pyogenes, E. coliinduced sepsis and meningococcal meningitis induced by Neisseriameningitidis.

[0215] The T cell system is far more adept at responding to innatepattern recognition units than to tumor associated antigens. In thepresent invention, tumor cells are transfected with nucleic acidsencoding molecules or biosynthetic enzymes that result in structureswhich mimic the major immunogenic structures of bacterial antigens. Thisenables the tumor cells to be recognized more effectively by the T cellsystem. In addition, tumor cells are provided with receptors forbacterial antigens such as SAgs, LPS's (CD14), and glycosylceramides(CD1). Genes encoding bacterial antigens which produce potent immuneresponses are transfected into tumor cells to include bacterial membraneand cell wall constituents such as LPS's, peptidoglycans,glycosylceramides, lipoproteins, lipoarabinans and capsularpolysaccharides. In addition, nucleic acids encoding the staphylococcalSAgs induce potent T cell lymphoproliferation and TH-1 cytokineproduction while LPS's are known to have a bystander effect on T cellproliferation The two agents synergize in their capacity to inducelethal endotoxic shock in animals.

[0216] The present invention contemplates that the optimal approach isto present the bacterial immunogen structure (for example streptococcalcapsular polysaccharide) sequentially or concomitantly with a bacterialmitogenic signal (SAg). Under certain conditions, these genes areco-transfected with various bacterial invasins, toxins, autolysins andinflammatory enzymes which together with the colonizing properties oftumor metastasis genes produce a tumor cell capable of migrating tometastatic sites where it induces necrotizing cellulitis. Such genes arepreferably placed under the control of inducible promoters as describedherein. These transfectants are prepared by methods in Example 1-3. Theyare useful against established tumors or metastatic tumor in vivo as inExample 15, 16, 18-23.

21b. Combining Expression of SAg Nucleic Acids with Nucleic AcidsEncoding Enzymes that Drive the Synthesis of Bacterial LPS,Galactosylceramide or Capsular Polysaccharide

[0217] In general, this is accomplished by co-transfection of nucleicacids each encoding one of the above products or by transfection with afusion nucleic acid that encodes the combination SAg-encoding nucleicacid is fused in frame or cotransfected into tumor cells or accessorycells with nucleic acids encoding bacterial LPS's, peptidoglycans, andgalactosylceramides. The preferred end products are synthesized in E.coli and N. meningitides (LPS's), Staphylococcus and Streptococcus(peptidoglycans); Sphingomonas paucimobilis (glycosylceramides).

[0218] The synthetic genome or cluster of genes for biosynthesis ofthese products is incorporated as a whole to include multiple andspecific enzymatic transferases and trafficking proteins required forthe stepwise synthesis of each of these products. Gene clusters arenecessary to provide the requisite transferases for synthesis of theselarge molecules. For example the genes required for the biosynthesis oftype 1 capsular polysaccharide of S. aureus are localized to a 14.6-kbregion. Sequencing analysis of the 14.6-kb fragment revealed 13 openreading frames (ORFs). Ten genes are involved in capsule biosynthesis.CapG aligned well with consensus sequence of a family ofacetyltransferases from various prokaryotic organisms suggesting thatCapG may be an acetyltransferase.

[0219] The structural requirements for endotoxic activity of LPS's areas follows. (1) a b(1-6)-linked D-glucosamine disaccharide backbone; (2)biphosphorylation at positions 1 and 4′ of the disaccharide backbone;(3) a suitable number of 3-acyloxyacyl groups per disaccharide unit; and(4) acyl groups of a suitable length as indicated by Kumazawa et al.,and Nakatsuka et al. Transfection with nucleic acid encoding LPS's wouldrequire the preservation of the biphosphorylation and the acyl groupsbetween 14 and 23 to maintain optimal activity. Derivatives may containa monosaccharide group in place of the disaccharide group.

[0220] LPS Structure

[0221] LPS consists of an outer region which is composed of polymerizeddi- and pentasaccharide repeating units whose compositions vary within aspecies or strain. The inner region is generally conserved within asingle genus, and consists of a core oligosaccharide linked by the sugar2-kcto-3-deoxy-D-amino-octonate (KDO to a disaccharide backbone withattached long chain fatty acids, the lipid A. This component isresponsible for much of the biological activity of the molecule.Components conferring the greatest biological and immunomodulatoryactivity are now known to be a glucosamine disaccharide, a bisphosphorylated lipid A and acyloxyacyl groups on the fatty acid chain.The loss of only one of these components, for example, a phosphate groupreduces the activity of the molecule. LPS's from different genera ofbacteria vary in their immunomodulating activity and studies of thestructure have shown very subtle differences. For example, Bacteroidesis apparently less active in endotoxin activity than LPS from entericbacteria. This was initially thought to be related to a modification ofthe of KDO in the core region with an added phosphate group. Otherdifferences in the LPS were found when the fatty acids from E. coli andBacteroides were compared. E. coli has six fatty acid chains or acylgroups per diglucosamine backbone each with a chain length of 12-14carbon atoms. Included in the acyl groups is 3-hydroxytetradecanoic acid(3-OH—C14:0)which is absent in the Bacteroides strains. In contrast,Bacteroides has 4-5 fatty acids of chain length 15-17 carbons perdiglucosamine and has branched 3-hydroxy fatty acids. Studies ofsynthetic lipids have confirmed that reduced biological activity relatesto fewer fatty acids chains.

[0222] A common feature of LPS's from various species is that they areamphiphiles, with both a hydrophobic part capable of dissolving in lipidmembranes and a hydrophilic part which remains in the water phase.Therefore, the first step of molecular interaction is one between theamphiphilic molecule and the mammalian cell surface either by ionicbinding, hydrogen bonding or hydrophobic interaction. The bacterialmolecule may be inserted into the mammalian membrane by its hydrophobicmoiety or attached to membrane receptors with the hydrophilic moiety, orthrough charge effects or via binding to host glycoproteins andglycolipids resulting in signal transduction. Most of theimmunomodulating activity of these bacterial molecules is indirect andstems from the release of host mediators. Cytokines such as IL-1, tumornecrosis factor, and IL-6 are involved. The LPS binding protein attachesto gram-negative bacteria or free LPS and mediates the attachment tomacrophage membrane receptor known as CD14. The recognition of the CD14only recognizes LPB when it is bound to LPS. The LPS-LPB complex maydirectly trigger TNF release or hold the complex at the cell surface sothat other hosts cell surface molecules trigger TNF release. LPBs alsoact as opsonins. Another area where sugar residues play an importantrole is in cell surface glycoprotein interactions which involveprotein-carbohydrate recognition. In the recirculation of andrecruitment of leukocytes in the body, the carbohydrate-recognizingprotein domains of glycoproteins of one cells bind specifically to theoligosaccharides of glycoconjugates on another type of cell. Theserecognition events control the movement of bloodborne lymphocytes intolymphoid organs. Specific recognition occurs between lymphocytes andspecialized cells in the wall of blood vessels known as high endothelialvenules.

[0223] Genes Encoding Lipid A Biosynthesis

[0224] LPS is generally synthesized as two separate components, thelipid A/core and the O polysaccharide, which are then ligated to givethe complete LPS molecule. Three genes encode enzymes that catalyze thesteps of lipid A synthesis (lpxA, lpxD and lpx B for steps 1,3 and 5)and fabZ and envA. More specifically, the enzymes that catalyze thesynthesis of lipid A are thought to act in the following sequence(indicating the genes): lpx A, lpx C, lpx D, lpxB. The reactionscatalyzed by the products of these genes are given in Table 1 ofSchnaitman C A et al., Microbiol. Rev. 57: 655-682 (1993).

[0225] Blocks of Genes Involved in LPS Biosynthesis

[0226] Blocks of genes involving LPS synthesis have been sequenced andanalyzed. The lipid A biosynthetic pathway has been elucidated. Four ofthe genes in this pathway have now been identified. Three of them arelocated in a complete operon which also contains genes involved in DNAand phospholipid synthesis. Genes involved in synthesis of the LPS lipidA core and their activity at various points in the biosynthetic pathwayare described in Schnaitman C A et al., Microbiological, Reviews 57:655-682 (1993) which is incorporated by reference. Therefore, it islikely that LPS biosynthetic enzymes are organized into clusters on theinner surface of the cytoplasmic membrane around a few key membraneproteins.

[0227] A cluster of assembly genes produced by various bacteria encodeLPS with homologous structures. These genes have been transfected intoE. coli and they induce identifiable LPS's. There are also smooth andrough LPS's which have a hierarchy of potency in terms of procoagulantactivity and activation of TNF. Mutants produced which synthesizedprogressively less polysaccharide attached to the lipid A moiety. Thepresence of long chain polysaccharides attached to the lipid moietydecreased the ability to activate TNF. Rough bacteria were moreeffective than smooth bacteria, in inducing TNF production. Fatty acidsof various chain lengths can be produced including those that resemblemonogalactosylceramides. Transferases for biosynthesis of galactan theLPS structure of the O antigen from Klebsiella pneumoniae have beenidentified as well as genes controlling the O antigen chain length.

[0228] The genes for LPS and glycosylceramide assembly also involvemultiple transferases. The transfection of tumor cells involves 10 genesencoding a particular stretch of the bacterial genome. In E. coli, the14-kilobase pair chromosomal region located between waaC (formerly rfaC)and waaA (kdtA) contains genes encoding enzymes required for thesynthesis and of the type R2 core oligosaccharide in the lumen of theendoplasmic reticulum. This occurs in a stepwise fashion.

[0229] The gene encoding the Haemophilus influenzae type B outermembrane protein functions as a porin and is useful in protectiveimmunity has been cloned as a 10-kilobase Hib DNA insert and expressedin E. coli. The biosynthesis of LPS's involves genes encoding the keytransferases including rfal. The N. meningitides highly conservedsurface protein conferring protection is encoded by a ORF of 525nucleotides.

[0230] Genes Encoding Enzymes the Catalyze Core Biosynthesis

[0231] The rfa cluster includes the genes for all transferases forassembly of core. It includes three operons consists of at least 17genes. The majority of known genes whose functions are involvedexclusively in LPS core biosynthesis are located in the rfa cluster[Pradel E et al., J. Bacteriology 174: 4736-4745 (1992)]. It includesthree operons. However, there are also genes such as kdsA and rfaElocated outside the rfa cluster which are involved in biosynthesis ofsugars unique to the core or exert direct effect on core structure.These clusters appear to have originated by the exchange of blocks ofgenes among ancestral organisms. There are few which code for theintegral membrane proteins. The promoter for the rfa genes has beenidentified. Mutations have been identified known a rough mutants tracedto three loci namely rfa, rfb and rfc.

[0232] The region of the E. coli chromosome encoding enzymes responsiblefor the synthesis of the LPS core has been cloned. This region formerlyknown as the rfa locus comprises 18 kb of DNA between the markers tdhand rpmBG. The genes are arranged in three different operons and thegenetic organization of this locus seems to be identical in E. coli K-12and S typhimurium.

[0233] Linkage of LPS Transcription and Toxin Secretion

[0234] In E. coli and Salmonella, a link has been found between toxinsecretion and the gene regulating

[0235] LPS transcription. Toxin secretion is regulated by geneexpression within the hlyCABD operon. A recently identified activator ofhlyCABD gene expression is the 128-kDa product of the rfaH (sfrB) genewhich positively regulates transcript initiation and possiblytermination in the operons encoding synthesis of LPS of E. coli andSalmonella. The discovery of a role in hlyCABD expression for the LPS(rfa) operon transcriptional activator rfaH is consistent with the roleof LPS in influencing both the secretion and toxic activity of thetoxin.

[0236] Genes Encoding Enzymes that Synthesize Polysaccharide Capsule andMembrane Proteins

[0237] Genes for the biosynthesis of a polysaccharide capsule areinduced in Sphingomonas by overlapping DNA segments which span about 50kbp restored the synthesis of sphingan. The polysaccharide components ofLPS from B. Pertussis, H. influenzae and Bacteroides spp. will activateB-cells. The polysaccharide of Bacteroides activates B cells indirectlyby first triggering the macrophage whereas the lipid A moiety triggersthe B cells directly. Therefore different parts of the same moleculeinteract with different types of host cells. There is also evidence thatimmunopotentiating activity of a glycopeptide produced by mycobacteriais dependent on the saccharide residues of the molecule.

[0238] The capsular polysaccharide of the Streptococcus is extremelyimmunogenic, consisting of glycan strands composed of regularlyalternating

[0239] N-acetylglucosamine and N-acetylmuramic acid residues joinedthrough β-1,4 glycosidic linkages and attached to crosslinked peptidesby amide bonds. The capsule of strain M is composed oftaurine-2-acetamido-2-deoxyfucose and 2acetamido-2-deoxy-D-galacturonicacid. The gene for this structure called cap-1 has been cloned and isused to transfect tumor cells. The nucleic acid sequences appear in Linet al., J. Bacteriol. 176, 7005-7016 (1994).

[0240] A new 24-kDa group A streptococcal membrane protein known asstreptococcal protective antigen (Spa) has been identified and isdistinct from the surface M protein which evokes protective opsonizingantibodies. The Spa-encoding gene has been cloned and consists of a636-bp 5′ fragment. (Dale, J B et al., J. Clin. Invest. 103: 1261-67(1999)).

[0241] The present invention contemplates the use for cancer treatmentof these and other bacterial antigens from staphylococci, streptococci,E. coli, N. meningitides, and other genera which antigens evoke animmune response in mammals. In the preferred approach, a nucleic acidencoding such an antigenic structure is transfected and expressed intumor cells. Methods of preparation, use and assessment of thesetherapeutic constructs in tumor bearing hosts are in Example 1, 2,18-23.

[0242] SAg nucleic acids are fused in frame or cotransfected into tumorcells or accessory cells with nucleic acids encoding key transferases(gene clusters) and glycosylation sites encoding capsular membrane fromStreptococcus or Neisseria meningitides lipoprotein-LPS-phospholipid andcell wall peptidoglycans, i.e., N-acetylglucosamine (NAG) andN-acetylmuramic acid (NAM).

[0243] SAg DNA is fused in frame to DNA encoding a highly conservedouter membrane surface protein of N. meningitides known as Nspa. TheNspa gene has been cloned (Martin, D. et al., J. Exp. Med. 185:1173-1183 (1997)). The LPS produced would be of weak to intermediatestrength such as that produced by Listeria or Legionella.

[0244] Borrelia burgdorfi is the causative agent of Lyme disease. Theosp genes are located at a single genetic locus on a 49 kbdouble-stranded DNA linear plasmid where they are organized as an operonospAB. The amino acid sequences of OspA and OspB show a high degree ofsimilarity and resemble prokaryotic lipoproteins. Nucleic acids encodingthe ospA and ospB lipoproteins are cotransfected into tumor cellstogether with SAgs.

[0245] Genes Encoding Membrane Glycosylceramide Biosynthesis

[0246] Nucleic acids encoding the synthesis of the GalCer fromSphingomonas paucimobilis are transfected into tumor cells, resulting inthe synthesis of GalCer by the tumor cell. (Kawahara K et al., FEBSLetters 292: 107-110, (1991) Yamazaki M et al., J. Bacteriology 178:2676-2687 (1996) Natori T et al., Tetrahed Lett 34:5591-5592 (1993)Costantino V et al., Liebigs Ann. Chem. 96: 1471-1475 (1995)). Nucleicacids encoding enzymes responsible for synthesis of Neiseriameningitides LPS are transfected into tumor cells, resulting in thesynthesis of LPS by the tumor cell (Steeghs L et al., Gene 190: 263-270(1997)). These nucleic acids encoding key transferases are fused tonucleic acids encoding amplified oncogenes or transcription factors suchas Bc1-2, c-myc, K ras, bcr, c-abl or NF-κB.

[0247] Genes Involved in Mycobacterial Cell Wall Biosynthesis

[0248] SAg-encoding nucleic acid is fused in frame or cotransfected intotumor cell with nucleic acids encoding the key enzymes involved in thebiosynthesis of mycobacterial cell wall mycolic acid,phosphatidylinositol mannosides and lipoarabinans. A high affinityinteraction of CD1b molecules with the acyl side chains of known T cellantigens such as lipoarabinomannan, phosphatidylinositol mannoside andmonomycolate has been demonstrated. Hence the nucleic acid encoding theCD1 receptor are cotransfected into tumor cells together withSAg-encoding nucleic acid and nucleic acids encoding the multifunctionalfatty acid and mycocerosic acid synthases involved in the biosynthesisof mycolic acid and methyl-branched fatty acids.

[0249] The multifunctional genes for mycocerosic acid synthase involvedin the biosynthesis of these molecules have been isolated. In additionto the usual fatty acids found in membrane lipids, mycobacteria have awide variety of very long-chain saturated (C18-C32) and monounsaturated(up to C26) n-fatty acids. The occurrence of α-alkyl β-hydroxy very longchain fatty acids i.e., mycolic acid is a hallmark of mycobacteria andrelated species. Mycobacterial mycolic acids are the largest (C70-C90)with the largest-branch (C20-C25). The main chain contains one or twodouble bonds, cyclopropane rings, epoxy groups, methoxy groups, ketogroups or methyl branches. Such acid are the major components of thecell wall, occurring mostly esterified in clusters of four on theterminal hexa-arabinofuranosyl units of the major cell wallpolysaccharides called arabinogalactans. They are also found esterifiedto the 6 and 6′ positions of trehalose to form “cord factor”. Smallamounts of mycolate are also found esterified to glycerol or sugars suchas trehalose, glucose and fructose depending on the sugars present inthe culture medium. Mycobacterium also contains several methyl-branchedfatty acids. These include 10-methyl C18 fatty acid (tuberculostearicacid found esterified in phosphatidyl inositide mannosides),2,4-dimethyl C14 acid and mono-, di- and trimethyl-branched C14 to C25fatty acids found in trehalose-containing lipo-oligosaccharides,trimethyl unsaturated C27 acid (phthienoic acid), tetra-methyl-branchedC28-C32 fatty acids (mycocerosic acids) and shorter homologues found inphenolic glycolipids and phthiocerol esters and multiple-methyl-branchedphthilceranic acids such as heptamethyl-branched C37 acid and oxygenatedmultiple methyl-branched acids such as17-hydroxy-2,4,6,8,10,12,14,16,-octamethyl C40 acid found insulfolipids.

[0250] Genes Involved in Mycolic Acid Biosynthesis

[0251] The biosynthesis of mycolic acids involves fatty acid chainelongation, desaturation, cyclopropanation of the olefin and aClaissen-type condensation. The genes involved in cyclopropanation arecma1, cma2. The methoxymycolate series found in M. tuberculosis containsa methoxy group adjacent to the methyl branch, in addition to thecyclopropane in the proximal position. A series of four methyltransferase genes was cloned. The mm4 methylates the distal olefin.

[0252] The multifunctional fatty acid synthase (FAS) (type 1) catalysesnot only the synthesis of C16 and C18 fatty acids but also elongation toproduces C24 and C25 fatty acids. Cloning and sequencing of the synthasegene revealed a 8389 bp ORF. The domain organization is much like a headto tail fusion of the two yeast FAS subunits; acyl transferase(AT)-enoyl reductase (ER)-dehydratase (DH)-malonyl/palmitoyltransferase-acyl carrier protein (ACP) fused with β-ketoreductase(KR)-β-ketoacyl synthase (KS).

[0253] The MAS gene encoding mycobacterial mycocerosic acid synthase isa dimer of the FAS gene. The cloning and sequencing of the MAS generevealed the domain organization in the following order: KS-AT-DH-ER-KR-ACP. The purified MAS shows a preference for elongation by fourmethylmalonyl CoA units reflecting the natural composition ofmycocerosic acids. FAS and MAS are also involved in the biosynthesis ofphthiocerol and phenolphthiocerol which involve elongation of preformedn-C20 fatty acyl chains or an acyl chain containing a phenol residue atthe w-end. The cluster of five genes, ppsi1-5 encode the multifunctionalenzymes (Fernandes N D et al., Gene 170: 95-99 (1996) Mathur M et al.,J. Biol. Chem. 267:19388-19395 (1992) Yuan Y. et al., Proc. Natl Acad.Sci. USA 92: 6630-6634 (1995)).

[0254] Tumor cells are cotransfected with SAg-encoding DNA and nucleicacids encoding the biosynthesis the above microbial products. Thetransfected tumor cells acquire significant additional immunogenicity.These cells are prepared as in Example 1-3. They are useful in vivo as apreventative or therapeutic antitumor vaccine (Examples 5, 15, 16 18-23.They are also useful ex vivo to immunize T, γ/δT cells, NK or NKT cellsto produce a population of effector T or NKT cells for adoptiveimmunotherapy of cancer (Examples 2-5. 15, 16, 18-23, 68).

22. SAg-Ganglioside or SAg-Galactosylceramide Complexes Formed afterTransfection of Tumor Cells with DNA Encoding SAgs: Complete BacterialAntigen System Recognized by CD1 Receptors Capable of InducingAnti-Tumor Effects

[0255] SAg-encoding nucleic acid transfected into tumor cells expressSAg on the tumor cell surface which is bound to cell surfacegangliosides which are tumor associated antigens, oncogene product suchas EGF or IGF. In this way the tumor associated antigen is capable ofrecognition and interaction with host T cells and macrophages and ofevoking a potent immune response. The SAg is also bound or associatedwith the CD1 receptor alone or associated with the glycosphingolipidtumor associated antigen.

[0256] SAgs have a natural affinity for glycosphingolipids on cellmembranes. Enterotoxin-producing-bacteria secrete enterotoxins which intheir precursor state are bound to cell membranes in dimeric form.Enterotoxin transfected tumor cells induce an anti-tumor response byexpressing the tumor cell surface antigen in association with the SAg.Bound to the tumor cell membrane, the SAg may be in dimeric formassociated with the ceramide lipophilic anchor domain of aglycosphingolipid tumor associated antigen. Likewise, the SAg may beassociated with the carbohydrate moiety or the ganglioside whichprotrudes from the cell surface. It may also be secreted in monomeric ordimeric form fused to membrane associated tumor antigen, oncogeneproduct or receptor. If the tumor associated glycosylceramide,glycoprotein antigens, or glycolipid antigen with or without SAg arepresented on CD1 receptors, then NKT cells may generate the predominantT cell response. However the classical T cell system is also responsive.

[0257] These constructs are produced and used as a vaccine againstestablished tumor by protocols given in Examples 2-5, 15, 16, 18-23.

23. Nucleic Acids Encoding CD1 Receptors

[0258] Nucleic acid encoding the CD1 receptor is transfected into tumorcells, resulting in expression of the CD1 receptor on the tumor cellsurface. Promoters of CD1 synthesis are also useful in this invention.The human genome includes five CD1 genes (A-D)which also function inantigen presentation to T cells (Calabi, F et al., CD1: From Structureto Function in Immunogenetics of the Major Histocompatibility Complex,Srivastava, R et al., eds, VCH publishers, New York, N.Y., 1991). Inmice, two homologous proteins (mCD1.1 and 1.2) have been characterizedand map to chromosome 3. The human CD1 genes are located on chromosome1q221-q23 in the order D-A-C-E from the centromere on a 190 kb segmentof DNA. With the exception of CD1B, they are all in the sametranscriptional orientation. They are evenly spaced in the complex withone exception: CD1D and CD1A are spaced two to three times farther apartthan the average. The products of CD1A, -B and -C genes have beendefined serologically. The products of CD1D and CD1E are unknown. Theyshare a highly conserved exon domain which is homologous to theβ2m-binding domain (α3) of MHC class I antigens. The CD1 molecules arenot polymorphic and apart from CD1D, are noncovalently associated withβ2m in a TAP-independent manner. Complex alternative splicing of CD1genes results in tissue specific forms of the protein, which can beintracellular, membrane bound, or secreted. In cells infected withmycobacteria, the CD1 molecule binds and presents a mycobacterialmembrane component, mycolic acid. Surface CD1 molecules present longerpeptides than those normally found on class I molecules. Whether CD1molecules can also present peptide antigens is still unclear althoughthis has been shown for at least one member of the CD1 family.

[0259] Tumor cells are transfected with nucleic acid encoding the CD1receptor. Nucleic acid encoding cell wall or cell membrane associatedglycosylceramides or a branched, b hydroxy long-chain fatty acids foundin mycobacteria and other bacteria are cotransfected into the CD1transfected tumor cells. The tumor cell therefore displaysglycosylceramides bound to the CD1 receptor.

[0260] Using site directed mutagenesis, DNA encoding the CD1 receptor isprovided along with DNA encoding a SAg binding site. This SAg bindingsite consists of key amino acids from the SAg receptor or from the SAgbinding sites on (i) MHC class II chains or (ii) the TCR Vβ region. Thismay consist of a glycosphingolipid sequence (sensitive toendoglycoceramidase) present on some mammalian cells.

[0261] The glycosylceramide used to bind to the CD1 receptor will havean exposed SAg binding site which is sensitive to endoglycoceramidase,an enzyme from Rhodococcus which specifically cleaves the glycosylmoiety from glycosphingolipids. Other ceramidases break up sphingolipidinto fatty acids and sphingosine.

[0262] These tumor cells transfectants are prepared as in Examples 1 and2. They are used in vivo as a preventative or therapeutic antitumorvaccine as in Example 14-16, 18-23. They are also useful ex vivo toproduce a population of tumor specific T, γ/βT, NK or NKT cells foradoptive immunotherapy of cancer (Example 2-5, 7, 15, 16, 18-23, 68).

24. DNA Encoding Streptococcal M Proteins and DNA Encoding Protein A orits Fc and VH₃ IgG binding Domains Transfected into Tumor Cells Alone orSAg DNA

[0263] The streptococcal M proteins are type-specific and act asprotective or virulence factors. M protein genes are members of a largeremm-like gene family, such that many S. pyogenes strains express morethan one M-like protein. DNA encoding the streptococcal M protein andDNA of the larger emm-like family are transfected into tumor cells(Kehoe M. A., “Cell-Wall Associated Proteins in Gram-Positive Bacteria,”In: Bacterial Cell Wall, Ghuysen J M et al., eds, Elsevier, Amsterdam,1994).

[0264] In addition, DNA encoding protein A and its domains as well asDNA of the streptococcal fcrΛ76 gene located upstream of the emm-likegene are transfected into tumor cells individually or together to causethe expression of IgG FcR- and VH3 IgG-binding domains (Kehoe Mass.,supra). DNA encoding SAg is cotransfected into the same tumor cells toproduce a tumor cell expressing any combination of M protein, protein Aand a SAg.

[0265] Such cells are used in vivo as preventive vaccines or astherapeutic vaccines against established tumors. See Examples 1-5, 11,15, 16, 18-23. They may also be used ex vivo to induce populations ofactive tumor specific effector T cells (including αβ+ T cells, γ/δ Tcells, NK cells and NKT cells) that are then used in adoptiveimmunotherapy See Examples 2-5, 7, 15-16, 18-23, 25, 68.

[0266] Nucleic Acids, Bacterial Cells and Phage Displays Mimicking SAgs

[0267] Because of circulating naturally occurring antibodies in humans,native or mutated SAgs that are administered parenterally are not likelyto reach the appropriate receptors on T cells or tumor cells. To solvethis problem, mimic oligonucleotides are prepared which mimic SAgs intheir capacity to bind SAg receptors. Since no natural antibodies aredirected to these compositions, they will not be prevented from reachingspecific SAg receptors in vivo.

[0268] SAg receptors are used to screen oligonucleotides for theirability to mimic SAg binding. Useful receptors for such screeninginclude those described herein (as expressed on tumor cells) and T cellTCR V chains. For example, pools of oligonucleotides are tested fortheir binding to, and affinity for, immobilized SAg receptors usingnucleotide chromatography technology well known in the art. Once thesehigh affinity binding oligonucleotides are identified, they are isolated(or, following sequencing, may be synthesized) and administered to ahost.

[0269] Also included here is a bifunctional oligonucleotide-peptidechimeric molecule that binds specifically to the SAg receptor on tumorcells as well as the Vβ region of the TCR. Such an oligonucleotide willbind simultaneously to tumor cells and T cells (in the process ofactivation) to produce an anti-tumor response. Anoligonucleotide-protein construct is prepared consisting of (a) apeptide sequence of enterotoxin A that binds to the TCR and (b) anoligonucleotide that binds to SAg receptor on tumor cells. The peptideportion of this construct should be devoid of MHC class II binding sitesin order to minimize undesired binding of the molecule to class IIstructures upon administration in vivo.

[0270] In another embodiment, the nucleic acid portion of the chimericmolecule binds to the TCR while the peptide consists of anon-enterotoxin ligand that is specific for the SAg receptor on tumorcells. This construct has the advantage of lacking any binding site fornatural antibodies. Yet another additional chimeric molecule consists ofan oligonucleotide portion specific for the class II α or β chain and asecond oligonucleotide or a peptide specific for the TCR Vβ chain.

[0271] Methods for preparing these constructs are given in Examples 5,13. These constructs are especially useful for targeting tumors in vivowhile also promoting a T cell anti-tumor response. See Examples 18-23.However, these chimeric molecules may also be used ex vivo in theproduction of tumor specific effector T cells capable of inducing, oreffecting, an anti-tumor response when administered to a tumor bearinghost. See protocols in Examples 2-5, 15, 16 18-23, 68.

[0272] SAg and GlycosylCeramide Co-Expression

[0273] This may be accomplished using intact bacteria or phage displayapproaches. Since the precursors and substrates of theglycosyltransferases are not readily available in most mammalian cells,it is more convenient to induce dual expression of GalCer and SAgs inbacteria, for example Sphingomonas paucimobilis, which naturally produceGalCer. Hence, nucleic acid encoding a SAg is transfected into thisbacterium together with a suitable promoter well known in the art. Thebacterium produces both GalCer and SAg.

[0274] By ensuring that the SAg contains one or more glycosylation sites(by using the appropriate nucleic acid sequence), a glycosylated SAg isproduced. Such a SAg binds to the glycosyl-ceramide, e.g., GalCer toform a conjugate that is expressed on the bacterial surface of issecreted. In either form, such a SAg-GalCer conjugate can sensitize NKTcells to produce an anti-tumor response.

[0275] In addition, phage or plasmids encoding the appropriatetransferase are transfected into low virulence Staphylococcus specieswhich also produce enterotoxins. The bacterium acquires the capabilityof expressing GalCer on its surface. These bacterial constructs andcompositions are used in vivo in a tumor bearing host to produce ananti-tumor response in protocols given in Examples 5, 13, 15, 16 18-23and Detailed Description Section 19. They are also are used ex vivo toactivate NKT cells or T cells to differentiate to tumor specificeffector cells for use in adoptive immunotherapy of cancer by protocolsin Example 1, 2, 14-16, 18-23).

[0276] Phage display technology is used to target selected SAg sequencesto targets in vivo. The selected peptide is used as a binding sequencesin lieu of the full-length polypeptide. This permits elimination fromthe construct of the antigenic portion of the SAg to which naturalantibodies are directed. Cloned genes are expressed as part of phagecoat proteins, for example, as fusions with the gene III protein (gIIIp)or the gene vIII protein (gVIIIp). In addition to the displayed geneproduct, the phage genome (of each particle) includes the gene encodingthis product.

[0277] Phage display is preferably done using the filamentous phagef88-4 and comprises forming a fusion that results in the C terminus ofthe “selected” (i.e., inserted gene's) product and the N terminus of thephage protein gVIIp. Peptides of various enterotoxins are expressed inthe phage display—most preferably peptides that bind to the SAg receptoron colon carcinoma cells. These peptides retain their capacity to bindto the TCR and to activate T cells. Also contemplated within thisinvention is phage display of SAg plus nucleic acid encoding synthesisof GalCer and/or the α-Gal epitope. DNA for synthesis of GalCer ispreferably isolated from Sphingomonas paucimobilis; DNA; encoding thegalactosyl transferase for synthesis of α-Gal is preferably isolatedfrom Klebsiella aerobacter, Serratia, E. coli and Salmonella organismswhich naturally produce and express these epitopes. The phage displaysare administered in vivo and are capable of initiating a potent immuneresponse to the tumor using the protocols described in Examples 5 and 13and Section 19, above. These preparations are also useful for activatingT cells or NKT cells ex vivo to produce a tumor specific effector cellsfor use in adoptive immunotherapy (Examples 2-5, 14-16, 18-23).

[0278] Viral infection of a host cell having the galactosyl transferaseresults in the shedding of virions that express the α-Gal epitope. Whena host mammalian cell has been transfected with nucleic acid encodingSAg, the virus can coexpress the Gal epitope and the SAg on its surface.Such a viral construct is administered in vivo to achieve a therapeuticeffect, or, in another embodiment, is employed ex vivo to produce tumorspecific effector T or NKT cells for use in adoptive immunotherapy ofcancer (Examples 2, 3, 7, 15, 16, 18-23).

26. Combining SAgs with Enterotoxin Precursors (Cell-bound Dimers andOligomers) and with Enterotoxin Promoters and Transcriptional RegulatoryGenes

[0279] Cell-bound SAg Dimers and Oligomers

[0280]Staphylococcal enterotoxins are present in the membrane ofenterotoxin producing bacteria in dimeric form and retain potententerotoxin-like activity when isolated from the membrane. It is in thismembrane-bound form that enterotoxins are combined with tumor associatedantigens or oncogene products and presented to the T cell system. Thedimerization of the enterotoxins may promote clustering for moreeffective presentation to T cells. Indeed, dimerization orpolymerization of enterotoxins or the introduction of tandem repeats ofthe SAg binding sites for TCR and MHC class II may be achieved by (1)site directed mutagenesis of the enterotoxin plasmid and (2)introduction of sequences for gene amplification, tandem repetitionand/or recombination or by (3) introduction of enzymes for peptide chainelongation. The duplication may be at the level of the bacterial operonincluding its transcriptional regulators, using methods well describedin the art.

[0281] Modified plasmid is DNA is introduced into the target tumor cellsor into accessory cells, either or both of which are useful in vivo as apreventative or therapeutic vaccine (Examples 1, 2, 15, 16, 18-23). Suchgenetically transformed cells may also be used ex vivo to produceeffector T or NKT cells for adoptive immunotherapy (Examples 1, 2, 7,15, 16, 18-23).

[0282] SAg agr locus (Accessory Gene Regulator) and other BacterialGenes and Elements

[0283] At least 15 gene coding for potential virulence factors in S.aureus are regulated by a putative multicomponent signal transductionsystem encoded by the agr/hld locus. The synthesis of at least 14exotoxins and enzymes in S. aureus is regulated by a set of trans-actingelements from agr. The agr gene coordinately controls the expression ofexfoliatin toxin, toxic shock syndrome toxin, a, b, d toxins,enterotoxin B, lipases and nucleases (Balaban, N. et al., Proc. Natl.Acad. Sci. USA. 92:1619-1623 (1995)). These proteins are members of thehistidine protein kinase family of regulators and control a number ofvirulence determinants (Balaban supra, Novick R P, Meth Enzymol. 204:587-637 (1991)).

[0284] Compared to wild-type, agr and hld mutants have decreasedsynthesis of extracellular toxins and enzymes (such as α-,β-, andγ-hemolysins, leucocidin lipase, hyaluronate lyase and proteases) whilehaving increased synthesis of coagulase and protein A. The agr geneconsists of two divergent transcriptional units driven by promotersnamed P2 and P3. The P2 transcript includes four open reading framesreferred to as agrA, B, C, and D, all four of which are required to forthe agr response.

[0285] The peptides predicted for agrA and agrC resemble the responseregulators and signal transducers of the two-component bacterial signaltransduction systems. The primary function of thee four genes discussedabove is to activate two promoters; the P3 transcript, RNAIII, howeveris the actual effector of the exotoxin response. RNAIII activatestranscription of secretory protein genes and represses transcriptions ofsurface protein genes. As a global regulatory system, agr, controls thepost-exponential production of exoproteins such as toxins, hemolysins,and exoenzymes. agr is a complex polycistronic locus that encodes atwo-component signal transduction pathway that activates transcriptionof a regulatory RNA molecule that in turn activates transcription of theexoprotein genes.

[0286] Thus, transcriptional regulation of the enterotoxin B gene aswell as SED, SEC and staphylococcal capsular polysaccharide geneinvolves the agr product. (agr does not regulate SEA expression).

[0287] The promoter region of SEA is localized by primer extensionanalysis. The 5′-end of SEA mRNA is localized 86 bp upstream of thetranslational initiation codon. A DNA region with good agreement withcanonical promoter sequences was observed beginning 8 base pairsupstream of the apparent transcriptional start site. No DNA upstream ofthe 35 bp region is required for transcription. Both the agr gene andthe SEA promoter have been cloned (Peng, H. L. et al., J. Bacteriol.170:4365-4372 (1988); Borst, D. W. et al., Infec. Immun. 61:5421-5425(1993)). The xpr locus and the agr locus interact at the genotypiclevel; agr is autoinduced by a proteinaceous factor produced andsecreted by the bacteria and is inhibited by a peptide from anexotoxin-deficient S. aureus mutant strain. The inhibitor, RIP, competeswith the activator, RAP. When given as a vaccine, RIP may prove usefulas a direct inhibitor of virulence.

[0288] A chromosomal locus (sar) distinct from agr, encodes aDNA-binding protein that is important in regulation, and is required forexpression of S. aureus exoproteins including enterotoxin, toxic shocksyndrome toxin, hemolysin and staphylokinase. Transcription of Protein Ais suppressed by sar and agr.

[0289] A list of plasmids containing bacterial virulence factors usefulin this invention is disclosed in Table 49, p. 223 of Patrick, S. etal., Immunological and Molecular Aspects of Bacterial Virulence, JohnWiley and Son, New York, N.Y. 1995.

[0290] This invention contemplates the use of the Staphylococcalenterotoxin promoters and transcription factors that activate theenterotoxin biosynthetic cycle. Several Staphylococcal promoters havebeen identified (Novick, supra). This invention also contemplates theuse of the peptide activator RAP which induces agr as well as thepeptide inhibitor RIP which induces or represses RNA III.

[0291] SAg-encoding nucleic acid is fused in-frame with Staphylococcusagr nucleic acid and introduced into tumor cells or accessory cells (orthe two are cotransfected into these cells). In another embodiment,SAg-encoding nucleic acids placed under the control of an enterotoxinpromoter, and this construct is introduced into tumor cells or accessorycells. The agr gene is especially useful because it can be linked to aninducible promoter such as that for corticosteroids or themetallothionein promoter, allowing it to be activated in a controlledmanner by exogenous administration of the inducing to the host.

[0292] Methods for introducing the above genes into tumor cells aredescribed in Example 1, 2, 11. The use of such cells in vivo aspreventative or therapeutic vaccines are discussed in Examples 15, 16,18-23. Use of these genetically transformed tumor cells ex vivo toinduce effector T, γ/δT, NK or NKT cells for adoptive immunotherapy isdescribed in Examples 2, 3, 7, 15, 16, 18-23, 68.

27. Combining SAg with Oncogenes, Protooncogenes, Amplified Oncogenes,Transcription Factors or Tumor Markers

[0293] In one embodiment, the nucleic acid encoding a SAg is fusedin-frame to oncogene or protooncogene nucleic acid in tumor cells oraccessory cells to produce a chimeric nucleic acid which is expressedin, or on the surface of, the cell. This fused gene may be renderedinducible by judicious choice of a promoter or other regulatorysequence. Preferably, such an inducible promoter is induced by a hormoneor a metal. A regulatory element, such as one activated by interferon ora cytokine (e.g., Jak or a STAT), may be included in this construct.

[0294] In another embodiment, the nucleic acid encoding SAg is fused inframe to nucleic acid encoding an oncogene which can be amplifiedmarkedly. The fused construct is introduced into tumor cells oraccessory cells. An amplified “unit” is initially much larger than thesize of the actual gene of importance to the oncogenic event(s)(Hellems, R E, Gene Amplification in Mammalian Cells, Marcel Dekker, NewYork, N.Y. ). Thus a silent gene is co-amplified with one or more genesexpressed on an amplicon. This is a preferred site for the insertinggene clusters wherein one gene encodes a SAg, others encode the enzymesof LPS lipid A biosynthesis, optionally together with their nativepromoters or operons.

[0295] Transcription Factors and Amplified Oncogenes

[0296] Oncogenes are frequently amplified in human tumors and culturedcancer cells. This is more characteristic of solid tumors and relativelyrare in lymphoid malignancies. DNA amplification was first observedcytogenetically a double minute chromosomes (DMs) or homogeneouslystaining regions (HSRs) but today, direct DNA analysis (Southernblotting) or molecular cytogenetic methodologies, such as fluorescencein situ hybridization (FISH) and comparative genomic hybridization (CGH)can be applied. DMs are episomal forms of amplified DNA that generallylack centromeres and are unequally distributed between daughter cells atmitosis. They appear as isodiametric extrachromosomal bodies stainablewith all chromatin dyes. HSRs are chromosomally integrated forms ofamplified DNA. They represent either the replacement of the normalchromosome banding pattern with an extended region of homogenousstaining or the insertion of such a region into an otherwise normallybanded chromosome. DMs and HSRs tend to be mutually exclusive and arepotentially interchangeable manifestations of the amplified DNA. Thus,DMs can potentially integrate into distant chromosomal sites to generateheritable HSR. Of 22 human tumors analyzed, 91% contained DMs only, 6.5%contained HSRs and 2.5% contained both. In solid tumors of epithelialorigin, DMs and HSR were found in 40% of breast carcinomas, 17% of nonsmall cell carcinoma of the lung, 18% of stomach and esophageal cancersand 15% of uterine carcinomas.

[0297] The overwhelming majority of oncogene amplifications in humantumors affect the Myc oncogene family. In small cell lung cancers allthree members of the Myc family, c-myc, N-myc and L-myc can be involved.Myc amplification is associated with a more invasive and more metastaticphenotype. N-myc amplification is seen in neuroblastoma and isassociated with the late stages and poor prognosis. The amplificationunits on chromosome 11q13 are seen in (a) breast cancer, (b) squamouscell carcinoma of the head and neck, lung, and esophagus and (c) bladdertumors. The amplification extends for over 1.5 megabase pairs of DNA andincludes two bona fide oncogenes: FGF3 and FGF4. It also includes theBc1-1 CCND1 (cyclin D1) as well as the EMS 1l gene that encodes thehuman homologue of cortactin. CCND1 has a critical role in amplified DNAsince its expression is increased as a consequence of amplification. Theother major targets for amplification are the genes encoding the EGFreceptor (ErbB1/Her1) and the related ErbB2/Her2. Both genes areamplified in breast cancer and other malignancies. ErbB2 is associatedwith estrogen receptor-negative breast cancers and poor prognosis.

[0298] Members of the myc gene family are activated in several humantumors as a result of DNA rearrangements through chromosomaltranslocations or gene amplification. When overexpressed, all myc genescomplement mutant c-ras oncogenes in the transformation of primary ratembryonic cells and transform Rat 1-A cells without assistance of otheroncogenes. Stimulation of cellular myc expression levels or changes inpost-translational modification of myc proteins have been followingexposure of cells to many growth promoting stimuli. These featuressuggest that the myc proteins participate in the final steps ofmitogenic signal transduction. The myc proteins act as transcriptionfactors involved in activation and/or repression of target genes. Inneuroblastoma, a group whose tumors are generally near diploid ortetraploid with chromosome 1p deletion(LOH) and N-myc amplification havea generally poor response to treatment and a poor prognosis. Genomicamplification of the N-myc cellular oncogene is present in approximately40% of cases of childhood neuroblastoma and correlates withhistopathological signs of advanced disease. This genomic N-mycamplification appears to be associated with tumor progression ratherthan tumor initiation since early stage tumors rarely exhibit M-mycgenomic amplification. Similarly the c-myc family of protooncogenesincluding N-myc and L-myc are amplified in small cell carcinoma of thelung.

[0299] The amplified oncogenes useful in the present invention includegenes encoding transcription factors. The preferred nucleic acids foruse in the present invention are c-myc, N-myc, c-abl, c-myb, c-crb,c-Ki-ras, N-ras. N-myc (amplified 5-1000

[0300] fold in neuroblastoma) is preferred. SAg-encoding nucleic acid iscotransfected into tumor cells or accessory cells with amplifiedoncogenes. The N-myc and L-myc genes have been cloned as c-mychomologous amplified oncogenes from human tumors. In one embodiment,SAg-encoding nucleic acid is fused in frame with nucleic acid encodingoncogenic transcription factors such as FOS, JUN. MYC, MYB and ETS. Inanother embodiment, such nucleic acid is cotransfected with SAg-encodingnucleic acids. Either of such constructs is introduced into tumor cellsor accessory cells. Proteins that interact with FOS and JUN are given inTable 1 p. 157 of Peters G et al., Oncogenes and Tumor Suppressors,Oxford University Press, Oxford UK 1997, incorporated by reference.

[0301] bcr/abl Gene

[0302] SAg-encoding nucleic acid is fused in frame or cotransfected withnucleic acids encoding the following agents and transfected into tumorcells and fused to oncogenic nucleic acids encoding chimeric proteinscapable of immunizing the tumor bearing host. An ideal candidates forsuch fusions is the bcr-abl gene which express the bcr/abl protein inchronic myelogenous leukemia (CML). The c-abl oncogene is amplified inchronic myelogenous leukemia. Scherle P A et al., Proc. Natl. Acad. Sci.USA 87: 1908-1917 (1990) Heisterkamp N et al., Nature 344: 251-253(1990). Abnormalities in the structure and expression of the human c-ablcellular oncogene have been associated with Philadelphiachromosome-positive CML which is present in more than 90% of cases. Thisaberrant chromosome marker is generated by a reciprocal translocationbetween chromosomes 9 and 22 in which the c-abl oncogene is translocatedfrom the distal end of the q arm of chromosome 9 to a relativelyrestricted 5-6 kb region on chromosome 22 termed the breakpoint clusterregion (bcr). This translocation creates a fusion gene that istranscribed as an 8 kb bcr-abl RNA that encodes the aberrant bcr-ablfusion protein product (P210) observed in CML cells. The bcr-abl fusionproduct has enhanced tyrosine kinase activity compared with the normalp145 c-abl product. Abnormalities in the structure and expression of thec-abl cellular oncogene have not been described in any type of humanmalignancy other than CML and Ph positive acute lymphatic leukemia. Geneamplification correlates with progression of malignancy.

[0303] EGF Receptor Genes

[0304] SAg-encoding nucleic acid is fused in frame to the nucleic acidsencoding the EGF receptor (EGFR) (Ulrich A et al., Nature 309: 418-421(1984)). The EGFR is the prototype of four-member receptor family. EGFRis frequently overexpressed or mutated in several different types ofhuman tumor. For instance, the EGFR is amplified in 20-40% of humanglioblastomas and a variety of epithelial tumors including head and necksquamous cell carcinomas, breast tumors, esophageal tumors andurogenital tumors. Amplification was accompanied by overexpression ofthe EGFR.

[0305] The erbB2 (her2/neu) Oncogene

[0306] SAg-encoding DNA is fused in frame to DNA encoding a tumor markersuch as PSA, c-erbB2(neu), her2/neu, bc1-2 and Brca-1. The principalamplified and functional genes in breast cancer are the growth factorreceptor-erbB2, the nuclear transcription factor c-myc, and the genesencoding cell cycle kinase regulatory genes termed cyclin D1 and cyclinEG. Gene amplification is thought to proceed via the initial formationof extrachromosomal, self replicating units (double minute chromosomes)that become permanently incorporated into chromosomal regions where theyare called homogeneously staining regions (HSRs) as described above.

[0307] The human counterpart of the oncogene neu known as her2 encodes aprotein of the same family as the EGFR. This family of genes has beencloned. Its products belong to a family of receptor tyrosine kinaseseach with a transmembrane domain, a cysteine-rich extracellular domainand an intracellular catalytic domain. They act as receptors for severalpeptide growth factors such as EGF, TGF(and neuregulins. The activatedreceptors are then able to bind to proteins containing src-homology-2(SH2) domains. The SH2 domain proteins recognize and bind to specificphosphotyrosine-containing sequences of the activated receptor. TheseSH2 containing adapter molecules, then trigger downstream signallingpathways, ultimately resulting in gene activation.

[0308] The erbB2 (neu/Her2) gene maps to chromosome 17p21 and codes fora 185 kDa transmembrane glycoprotein related to, but not identical tothe EGF receptor (Schechter A L et al., Science 229: 976-978 (1985),Bargmann C L Nature 319: 226-230 (1986), Hung M C et al., Proc. Natl.Acad. Sci. USA 83: 261-264 (1986), Yamamoto T et al., Nature 319:230-234 (1986)). The EGFR bears sequence homology with the erbB1product. The erbB2 gene is activated by a point mutation which mutatesamino acid residue 664 from valine to glutamic acid; this change isassociated with transforming its ability. The genes are called erbB,(erbB1, EGFr), erbB2, (neu/Her-2). erbB3(HER-3) and erb B4 (HER-4).

[0309] Amplification and overexpression of erbB2 has been found in avariety of human tumors including carcinomas of the breast, ovaries,colon, lung, liver, stomach, kidney, esophagus, salivary gland, andbladder. Genomic amplification of the neu (C-erb-2) or HER2 cellularoncogene and protein overexpression has been documented in approximately30% of primary human breast cancers and may correlate with advanceddisease and a relatively poor prognosis. More than 50% of all ductalcarcinomas in situ of the large cell type express HER2. Amplificationoccurs in approximately 20% of invasive breast carcinomas. Thus, it isthought that HER2 amplification increases the growth rate but not themetastatic potential of tumor cells.

[0310] A third member of the EGFR family is ERBB3which is present insome human breast cancers with high expression correlating with lymphnode metastases. Overexpression of ERBB3 has been observed in epidermoidcarcinoma of the larynx and esophageal carcinoma. ERBB4, a fourth memberof the EGFR family, was overexpressed in a human mammary tumor cellline. Fisk et al. (J. Exp. Med. 181: 2109-2117 (1995)) described animmunodominant epitope of HER/neu that is recognized by ovariantumor-specific cytotoxic T lymphocytes. This epitope is useful in thisinvention. Failure of coexpression of a heterodimeric partner orcoinduction of a suppressor phosphatase would explain the lack ofimmunogenicity of c-erbB2 in mice in nude mice.

[0311] Additional oncogenes, protooncogenes and tumor markers whichwould be candidates for the fusion in accordance with this invention arew PSA, c-erb B2(neu), Her2/neu, bc1-2, Brca-1. Viral and non-viraloncogenes and protooncogenes which are overexpressed in tumor cells areshown in Table 9.2 and 9.1, p. 171-172 of Franks et al., supra). Thefunctions of the various oncogenes is shown in Table 9.6, p.186, ofFranks et al.

[0312] IGF Receptor Genes

[0313] SAg-encoding nucleic acid is fused in frame with nucleic acidsencoding insulin-like growth factor (IGF) receptors (IGFRs)andtransfected into tumor cells. The IGFR gene is a tyrosine kinasecontaining transmembrane protein that plays an important role in cellgrowth control. There is a single IGF1 receptor gene with a completecoding sequence contained in 21 exons (Abbott A M et al., J. Biol. Chem.267: 10759-10763 (1992); Scott J et al., Nature 317: 260-262 (1985); LiuJ et al., Cell 75: 59-63 (1993)).

[0314] IGF1R is expressed at high levels in breast cancer, andamplification of the IGF1R gene has been observed. IGFs play asignificant auxiliary role in tumor growth by suppression of apoptosis.The apoptotic effect overexpressed myc is overcome by IGFs. Thus, IGFsfacilitate tumor growth by suppression of apoptosis.

[0315] Fibroblast Growth Factor (FGF) Receptor Genes

[0316] SAg-encoding nucleic acid is fused in frame to nucleic acidsencoding fibroblast growth factors receptors (FGFs) and transfected intotumor cells. FGF receptor are also be important for the vascularizationof certain types of tumors. The expression of FGF1 has been shown to beassociated with a switch to an angiogenic phenotype during thedevelopment of a fibrosarcoma. Overexpression of FGF receptor by certaintumors may also contribute to their growth. FGF receptors have beenshown to be amplified in some breast cancers.

[0317] Platelet Derived Growth Factor (PDGF) Receptor Genes

[0318] SAg-encoding nucleic acid is fused in frame to nucleic acidsencoding additional tumor growth factors which are produced oroverexpressed and transfected into tumor cells or accessory cells.Growth factors include those in the tyrosine kinase receptor familiessuch as Platelet Derived Growth Factor A and B family (PDGF). PDGF A andB receptors are amplified in malignant glioblastomas in the malignantcells themselves or the stromal cells (Fleming T P et al., Cancer Res.52: 4550-4556 (1992);Kumabe T et al., Oncogene 7: 627-632 (1992)). Thenerve growth factor (NGF), stem cell factor receptor (kit), colonystimulating factor-1 receptor (fins), neurotropin receptor family,transforming growth factor b family, the WNT family, angiogenicreceptors

[0319] Other Amplified Oncogenes

[0320] SAg-encoding nucleic acid is also fused to nucleic acid encodingthe tyrosine protein kinases which are both membrane associated andtransmembrane as described in Table 9.4, p. 179 of Franks et al., supra.Additional chromosomal regions which are amplified in greater than 40%of cases included the 8q24 locus of the c-myc(O) gene, the 11q13 locusof the cyclin D (O), int2 (O), EMS-1 (O), BCL-1 (O), FGF-4 (O) GST (M),MEN1(S) genes, the 17q21 locus of the RARa (S), RARg (S), ERBAa (S),BRCA1 (S), NM23 (S), estradiol 17B dehydrogenase (S) ERG2 (O), HOX2,NGFR (O), WNT3 (O) and the 20q13 locus.

[0321] Nucleic acid encoding SAg is fused or cotransfected into tumorcells with nucleic acid encoding the above oncogenes, amplifiedoncogenes and protooncogenes, transcription factors and growth factorreceptors. These transfectants are prepared as in Examples 1. They areuseful in vivo as a preventative or therapeutic vaccine (Examples 15,16, 18-23). They are also useful ex vivo for inducing tumor specificeffector cells (T cells, γ/δ T cells, NK cells, NKT cells) for adoptiveimmunotherapy (Examples 2-5, 7, 15, 16 18-23, 68).

28. Combining SAg with Angiogenic Receptors and Growth Factor Receptors

[0322] SAg-encoding nucleic acid is cotransfected or fused in frame tonucleic acid encoding an angiogenic receptor such as VEGF andtransfected into tumor cells. SAg nucleic acid is also fused to orcotransfected with nucleic acid encoding other angiogenic receptors suchas V integrin, other integrins, cadherins or selectins and introducedinto tumor cells or accessory cells. SAg-encoding nucleic acid is alsocotransfected into tumor cells or accessory cells with nucleic acidsencoding angiogenic proteins such as VEGF.

[0323] VEGF is produced by tumor cells and stroma, and its expressioncorrelates with the degree of vascularization and grade of malignancy.VEGF receptors, termed KDR and flt, are expressed mainly by the tumorendothelium. Higher levels of VEGF are found in metastatic than innon-metastatic colon cancers (Tischer E et al., J. Biol. Chem. 266:11947-11954 (1991). VEGF is especially useful here because it isoverexpressed in tumor cells at an early stage of tumorigenesis. Thepromoter of the VEGF gene lacks a TATA box, but has six GC boxes fortranscription factor SP-1 binding and also a site for AP-1 and AP-2binding.

[0324] The expression of the gene is modulated by several growth factorssuch as EGF. In some cell types VEGF expression is regulated by IL-1,FGF, PDGF. A common element, mediation of protein kinase C in theregulation of VEGF, has been suggested. VEGF is expressed as a disulfidelinked dimer. Long and short forms are generated by alternative splicingand are matrix bound or released, respectively. As a result of itsspecific effects on endothelial cell migration and proliferation, VEGFis a very potent and specific promoter of angiogenesis. Two wellcharacterized families of angiogenic factors act by binding to tyrosinekinase receptors that have two or three immunoglobulin-like domains, andVEGF binds to two related receptors with seven immunoglobulin-likeextracellular domains.

[0325] The TRKA oncogene codes for a receptor for nerve growth factor(NGF). The TRKA gene has been found fused to genes that code forproteins that form dimers in cells leading to the synthesis of aconstitutively dimerized and active tyrosine kinase. TRKA may have atumor suppressor function since its expression in neuroblastomacorrelated inversely with n-myc gene amplification. Coexpression of mRNAfor TRKA and the low affinity NGF receptor in neuroblastoma correlatedwith a favorable prognosis.

[0326] Nucleic acid encoding SAg is fused to nucleic acid encoding theabove angiogenic factors or receptors and introduced into tumor cells;alternatively, the two nucleic acids are used to cotransfected tumorcells. These transfectants are prepared as in Example 1. They are usefulin vivo as a preventative or therapeutic antitumor vaccine (Examples 15,16, 18-23). They are also useful ex vivo for inducing tumor specificeffector cells (T cells, γ/δT cells, NK cells, NKT cells) for adoptiveimmunotherapy of cancer (Examples 2-5, 7, 15, 16 18-23, 68).

29. Combination of SAg with Cell Cycle Protein

[0327] SAg-encoding nucleic acid is fused in frame to nucleic acidencoding a cell cycle protein such as a cyclin which is overexpressed intumor cells. Examples of these cell cycle proteins which are preferredfor such fusions are Cyclins A, B, D1, E. These proteins are generallycomplexed to kinases or transcription factors at critical checkpoints inthe cell cycle. The cyclins, CDKs and their inhibitors are shown inTable 1. p193 of Peters G et al., supra.

[0328] In another embodiment, nucleic acid encoding SAg is cotransfectedinto tumor cells with nucleic acid encoding a cell cycle protein asabove. These transfectants are prepared as in Examples 1. They areuseful in vivo as a preventative or therapeutic antitumor vaccines(Examples 15, 16, 18-23). They are also useful ex vivo for inducingtumor specific effector cells for adoptive immunotherapy of cancer(Examples 2-5, 7, 15.16 18-23, 68).

[0329] “Tumor specific effector cells” in this section and any otherplace in this application refer to T cells of any kind or type includingbut not limited to α/β+ T cell, αβ−T cells, γ/δT cells, NK cells, NKTcells. These cells may be used individually or collectively in anycombination for adoptive immunotherapy.

30. Combining SAg with Tumor Suppressor Genes, p53 or DevelopmentalGenes

[0330] SAg-encoding nucleic acid is fused in frame with tumor suppressorgene DNA and the fused nucleic acid is introduced into tumor cells oraccessory cells. Alternatively, the two nucleic acids are used tocotransfected these cells. Examples of such tumor suppressor genes areshown in Table 9.7 p.187 of Franks L M et al., supra. Examples ofmutated tumor suppressor genes include the APC and MCC genes and theirisoforms, the DCC gene in colon cancer, the BRCA1 tumor suppressor genein breast cancer and the DPC gene in pancreatic cancer. The p53 gene andits mutations are also useful in this embodiment. A list of p53responsive elements and associated proteins useful in this invention isgiven in Tables 1 and 2 pp. 267-269 of Peters G et al., supra.

[0331] In another embodiment, nucleic acid of developmental genes isused in place of tumor suppressor or p53 genes. Examples of suchdevelopmental or differentiation genes are wnt and fwt genes.

[0332] Transfectants are prepared as in Example 1. They are useful invivo as a preventative or therapeutic antitumor vaccine according toExamples 15, 16, 18-23). They are also useful ex vivo for inducing tumorspecific effector cells for adoptive immunotherapy of cancer (Examples2-5, 7, 15, 16, 18-23, 68).

31. Combining SAg with Cell Surface Glycoproteins or their Receptors

[0333] SAg-encoding nucleic acid is fused in frame with a nucleic acidencoding a cell surface glycoprotein and or its receptor and the fusednucleic acid is introduced into tumor cells or accessory cells.Alternatively, the two nucleic acids are used to cotransfected thesecells. Examples of these glycoproteins or receptors include integrins,vitronectin receptors, laminin receptors, cadherins, tenascin and CD44and isoforms, VCAM-1, P-Selectins, E-Selectin, NCAM and MCAM.Transfectants are prepared as in Example 1. They are useful in vivo as apreventative or therapeutic antitumor vaccine according to Examples 15,16, 18-23). They are also useful ex vivo for inducing tumor specificeffector cells for adoptive immunotherapy of cancer (Examples 2-5, 7,15, 16 18-23, 68).

32. Combining SAg with Cytokines and Chemokines and Chemokine Receptors

[0334] SAg-encoding nucleic acid is fused in frame with nucleic acidencoding a cytokines and chemokines, and the fused nucleic acid isintroduced into tumor cells or accessory cells. Alternatively, the twonucleic acids are used to cotransfected these cells. Examples ofchemokines and cytokines that are useful herein include RANTES, IL-5,IL-7, IL-12, IL-13, IFN-γ, TNF-α and TNF-β. Chemokines are small(typically 6-10 kDa) peptides that have been divided into two classesdesignated C—C and CXC based on the sequence of the first two cysteineresidues. The two families exhibit preferences for different target celltypes: C—C chemokines act primarily on macrophages.

[0335] Chemokine gene expression is induced by the action of othergrowth factors and cytokines and are actively expressed in solid tumorsshowing inflammatory involvement and macrophage or neutrophil invasion.Chemokines of the C—X—C class containing the amino acid, sequence motifELR have demonstrable angiogenic activity which can be inhibited byC—X—C chemokines lacking the ELR motif. Therefore chemokine expressionby either tumor cells themselves or elicited from stromal cells by theaction tumor-derived growth factors, have the potential to regulatetumor growth by modulation of angiogenesis. G-CSF is a growth factor forgranulocyte precursors, and IL-2 is a growth factor for T cells.

[0336] Nucleic acids encoding SAgs are fused or cotransfected into tumorcells with nucleic acids encoding the above cytokines, chemokines andchemoattractants. The transfectants are prepared as in Example 1. Theyare useful in vivo as a preventative or therapeutic antitumor vaccineaccording to Examples 15, 16, 18-23). They are also useful ex vivo forinducing tumor specific effector cells for adoptive immunotherapy ofcancer (Examples 2-5, 7, 15,16 18-23).

[0337] Chemokine Receptors: Previously we showed that 4T1 mammarycarcinoma cells transfected with superantigens and CD80 molecules couldbe used therapeutically to reduce the number of established lungmetastases and prolong survival in a murine model of post surgicalmammary carcinoma (Pulaski, B. A., Terman, D. S. et al., Cooperativityof Staphylococcal aureus Enterotoxin B Superantigen, MajorHistocompatibility Complex Class II, and CD80 for Immunotherapy ofAdvanced Spontaneous Breast Cancer in a Clinically RelevantPostoperative Mouse Breast Cancer Model, Cancer Research 60: 2710-2715(2000)). It is believed that the superantigen transfected tumor cells,administered parenterally to the tumor bearing hosts, traffic to organswhich are colonized by metastatic tumor cells. At these sites, the tumorcell transfectants secrete superantigen which induces a powerful local Tcell inflammatory tumoricidal response.

[0338] The present invention aims to enhance the targeting of thesuperantigen transfected tumor cells to metastatic sites. This isaccomplished by cotransfecting the tumor cells with nucleic acidsencoding superantigens and chemokine receptors including but not limitedto CXCR4. It has been shown previously that tumor cells expressing CXCR4will metastasize to organs with a natural abundance of the ligand forCXCR4. For breast carcinoma, this includes organs which are rich inCXCL12 the natural ligand for the CXCR4 receptor.

[0339] While the CXCR4/CXCL12 system is exemplified here, any otherchemokine receptor which is expressed in tumor cells and contributes tothe metastatic colonization of organs rich in its receptor ligand couldbe used.

[0340] The gene for the CXCR4 receptor has been identified (Wegner S Aet al., J. Biol. Chem. 273: 4754-4760 (1998); Caruz, A M et al., FEBSLett. 426: 271 (1998); Frodl, R. et al., J. Receptor Signal Tranduct.Res. 18: 321 (1998)). The DNA or RNA encoding the chemokine receptor anda superantigen are cotransfected into tumor cells by methods given inExample 1. The transfected cells are tested in the 4T1 model ofmetastatic carcinoma in BALB/c female mice as described by Pulaski andTerman et al., Cancer Res. 60: 2710-2715 (2000). Alternatively, the SCIDmouse model of established breast cancer using superantigen andchemokine receptor transfected into MDA-MB-231 human breast carcinomacells as the therapeutic agents described by (Muller A. et al, Nature410: 50-56 (2001) is used.

[0341] Superantigen and chemokine receptor transfected dendritic cells(using RNA or DNA encoding superantigens and chemokine receptors) areused to promote their migration and colonization of metastatic tumorsites. Once localized to tumor, the transfected dendritic cellsphagocytose tumor cells and present them together with expressedsuperantigen to the T cells which then effect a potent T cellinflammatory tumoricidal response. In addition to tumor cells anddendritic cells mentioned above, other antigen presenting cells,accessory cells such as fibroblasts and macrophages are useful.Epithelial cells or organ-specific cells which express chemokinereceptors are useful for transfection of superantigen nucleic acids.

[0342] The therapeutic system may comprise individual or mixedpopulations of tumor cells or accessory cells transfected with nucleicacids or RNA encoding superantigens and chemokine receptors as well ascostimulatory molecules such as CD80, and MHC class II. The optimaltherapeutic combinations consists of at least one population (at least50%) consisting of tumor or accessory cells cotransfected withsuperantigens and chemokine receptor(s) together with a second cellpopulation of tumor cells and/or accessory cells cotransfected with MHCclass II and CD80 or other costimulatory molecule. The epithelial cellsor organ specific cell types stipulated above maybe substituted for thetumor cells or accessory cells in this system.

[0343] The transfectants are administered parenterally by infusion orinjection according to the protocol of Pulaski, BA ; Terman D S et al.Cancer Res. 60: 2710-2715 (2000). The number of metastases in the lungsas well as survival of experimental and control ice are determined asgiven by Pulaski, B A, Terman, D S et al. Cancer Res. 60: 2710-2715(2000).

33. Combining SAg with Transcription Factors AP-1 and NFκA

[0344] Transcription factor genes may act as oncogenes. The jun familyof transcription factors bind specifically to AP-1 sites which conferthe effects of potent tumor promoting phorbol esters on responsive genesand specifically bind to c-jun homodimers or c jun/c-fos heterodimers.v-rel encodes members of the NF-kB family of transcription factors.Transforming oncogenes such as v-ets and v-myb also encode transcriptionfactors.

[0345] The T cell signaling system responding to SAgs activates the JAK,TNF (TRAF), IL-2 and IL-12 pathway probably via NFκA activation. LPS hasa T cell stimulating effect and may fuse with SAg to produce additionalstimulation or epitope expansion. The NFκA nucleic acids are fused to apromoter which activates sequences encoding the SAg receptor or thesequences encoding the key Vβ domains binding SAgs or regions in the Vβreceptor which are activated by the SAgs.

[0346] SAg-encoding nucleic acid is fused in frame with nucleic acidsencoding a transcription factor such as those above. Transfectants areprepared as in Example 1. These transfectants are prepared as inExample 1. They are used in vivo as a preventative or therapeuticantitumor vaccine according to Examples 15, 16, 18-23). They are alsoused ex vivo for inducing tumor specific effector cells for adoptiveimmunotherapy of cancer (Examples 2-5, 7, 15, 16 18-23, 68).

34. SAgs Augment the Immunostimulatory Effects of Tumor AssociatedPeptides, Binary and Ternary Complexes

[0347] Bacterial SAg are presented to T cells via the MHC class IImolecule by multiple low affinity attachments, resulting in stimulationof the T cell with very low concentrations of antigen. SAgs augment thepresentation of antigenic peptides to T cells without stericallyinterfering with each other's ability to bind and activate the TCR.These augmenting peptides are incorporated into the SAg structure. SAgsmay also bind to binary or ternary complexes of tumor peptide-MHC classI or tumor peptide-MHC class II complexes, either in solution or affixedto a TCR or the surface of an APC.

[0348] In one embodiment, the SAg is first bound to APCs or T cellsfollowed by addition of complexes between MHC class I or class II andtumor peptide. Alternatively, the SAg may first bind to eithercell-bound, soluble or immobilized MHC class I or class II molecules,after which the tumor peptide is added. This trimolecular complex isthen presented to the T cell via the TCR.

[0349] In another embodiment, SAg is first bound to an APC or to a TCRVβ chain on an NKT cell. Following this, CD1-glycosylceramide complexesare added and allowed to bind to NKT cell TCR Vβ chain. SAg may be boundto first to CD1-glycosylceramide complexes in soluble form, affixed toCD1+ cells or NKT cells via the TCR. SAgs may be bound to CD1 complexeswith glycosylceramide or a glycosphingolipid (with a conserved SAgbinding site) in solution or when fixed to CD1+ cells or NKT cells.Alternatively, SAgs are bound to ternary complexes consisting ofCD1-glycosylceramide affixed to the NKT cell TCR or bound toCD1-glycosylceramide on APCs, in solution or immobilized, before it hasaffixed to the NKT TCR. SAg is alternatively bound to binary complexesof (a) CD1-glycosylceramide, (b) CD1-glycosphingolipid, (c) CD14-LPS or(d) MHC-tumor peptide complexes that have either a SAg receptor sequenceor a TCR Vβ SAg-binding sequence.

[0350] The complexes described above are used in vivo as preventative ortherapeutic antitumor vaccines according to Examples 4, 15, 16, 18-23.They are also used ex vivo for inducing tumor specific effector cellsthat are then taken for adoptive immunotherapy of cancer. (See Examples2-5, 7, 14, 15, 16 18-23, 68).

35. SAgs Combined with Products of Antigen Processing Pathways

[0351] A chimeric gene is prepared consisting of SAg-encoding nucleicacid fused in frame to nucleic acids encoding (a) the endoplasmicreticulum (ER) translocation signal peptide, (b) transmembrane domain,and (c) lysosomal targeting domain of LAMP-1. LAMP-1 is a type 1transmembrane protein localized predominantly to lysosomes and lateendosomes. The cytoplasmic domain of LAMP-1 contains the Tyr-Gln-Thr-Ilesequence that mediates the targeting of LAMP-1 into the endosomal andlysosomal compartments. The specific targeting of the SAg to theendosomal and lysosomal compartments allows SAg peptides to complex withMHC class II molecules and enhance presentation.

[0352] The MHC class I presentation pathway operates on a three levelsystem. At one level there is protein machinery dedicated to peptidemanufacture—the proteosome complex. The selective peptide transportersdeliver antigens into the ER. The class I molecules themselves exhibitvariable affinities for peptides. Genes clustered in the region of theclass II gene encode proteosome and transporter. SAg peptides aretransported into the ER—primarily through a transmembrane “tube”consisting of two polypeptide chains called TAP-1 and TAP-2 (transporterassociated with antigen processing). In mammals, genes encoding TAP-1,TAP-2 and two proteosome polypeptides are all located within the classII region of the MHC.

[0353] The class I pathway starts in the cytosol where proteins producedinside the cell are degraded by the multicatalytic proteosome complex.The peptide products are translocated into the ER by the TAP proteins.In the lumen of the ER, the peptides bind the class I protein groovewhile the latter are complexed with the chaperone p88, P₂M and TAP.After securing a peptide in its binding groove, the class I complex isreleased from TAP and transported through the Golgi apparatus to thecell surface. TAP genes are closely linked to the LMP2 and LMP7 in theclass II MHC gene cluster and belong to a family of molecules involvedin ATP-dependent membrane translocation known as the ABC (ATP-bindingcassette) transporters. TAP1 and TAP2 function as a heterodimer eachsubunit having over 500 amino acids each with two hydrophobic domains,six membrane spanning regions and a cytosolic ATP binding motif BothTAP1 and TAP2 subunits are required for peptide binding andtranslocation. TAP1 appears to be uniquely involved in the interactionswith class I/β2 dimers at the luminal membrane of the ER where itinteracts with the membrane proximal region of the α3 domain of classI-β₂m complexes prior to peptide loading. Interaction between class Iand TAP is crucial for efficient peptide loading. Antigen presentationis mediated by an additional factor, tapasin. TAP also binds 2Mindependently of class I heavy chain, perhaps facilitating rapidassembly of class I peptide-binding complexes. TAP heterodimer may showa preference for amphipathic molecules as T cell antigenic determinantsare often seen clustered around sequences where amphipathic helicalstructures are predicted. TAP prefers peptides 8-10 residues in lengthbut may transport peptides ranging from 7-40 residues.

[0354] Invariant chains are transmembrane glycoproteins found inintracellular compartments in association with class II molecules.Multimers consisting of three class I ab dimers and three invariantchains assemble rapidly in the ER and travel across Golgi bodies to thetrans-Golgi network that intersects with the endocytic pathway, whereclass II molecules reside for about 1-3 hr before transit to the cellsurface for display to T cells. Alternative splicing of the invarianttranscripts produces two isoforms p31 and p41 both of which can operateto assist folding of class II dimers, direct the passage of class IIfrom the ER through an exocytic pathway, and block loading of peptideuntil peptide sampling can occur as exocytic-endocytic pathwaysintersect. A four residue targeting signal at the N-terminus of theinvariant chain that is essential for intracellular transport toendosomal compartments. The C-terminus and the transmembrane region orthe invariant chain are also necessary for sorting of class II-invariantchain complexes to the endosome. p41 appears to regulate the productionof a stable 12-kDa SLIP-class II complex capable of enhancing SAgpresentation.

[0355] SAg-encoding nucleic acid is fused in frame with nucleic acidencoding a protein involved in the antigen processing pathway such asthe invariant chain or TAP which facilitates the expression of the SAgin the context of MHC class I and II, respectively. Tumor cells,accessory cells and hybrids thereof are transfected with fusedSAg-invariant chain DNA as in Examples 1 and 5. They are used in vivo asa preventative or therapeutic antitumor vaccine according to Examples15, 16, 18-23. They are also used ex vivo for inducing tumor specificeffector cells for adoptive immunotherapy of cancer (Examples 2-5, 7,15, 16 18-23).)

[0356] SAg polypeptide post translationally is fused or associated withadditional molecules such as mono and diglycosylceramides, including butnot limited to—anomeric mono—and digalactosylceramides GalCer, α-gal,glycosylated and prenylated SAgs. These constructs translocate with theappropriate trafficking molecule e.g., invariant chain, TAP, LMP, toselected surface receptor such as MHC class I, MHC class II or CD1.These transfectants are prepared as in Example 1. They are useful invivo as a preventative or therapeutic antitumor vaccine according toExamples 15, 16, 18-23. They are also useful ex vivo for inducing tumorspecific effector cells for adoptive immunotherapy of cancer (Examples2-5, 7, 15.16 18-23, 68).

36. SAgs Combined with Signal Transduction Molecules or Heat ShockProteins (HSPs)

[0357] SAg-encoding nucleic acid is fused in frame to (or cotransfectedwith) a nucleic acid encoding “signal transduction molecules” such asRas, JAK 1 and STAT-1a and heat shock proteins HSP-60, HSP-70, HSP-90a,HSP-90b, Cox-2 as well as heterotrimeric G proteins and ATPases. Thegenes for Staphylococcal HSP-70 useful in this invention have beencloned (Ohta, T et al., J. Bacteriology 176: 4779-4783, (1994)). As usedherein, SAg polypeptides are ligated to any of above structures at thepeptide or nucleic acid level. Preferred proteins for this embodimentare G proteins, ATPases and HSPs. Chemical conjugation is carried out byconventional methods, e.g., use of preferred heterobifunctionalcrosslinkers. Alternatively, conjugates are produced genetically asfusion proteins by conventional methods. In yet another embodiment, theconjugates are created by permitting natural binding of the componentsto each other without chemical modification. Any of the foregoingconjugates or fusion proteins may be used when incorporated intovesicles or exosomes secreted from a cell. See Example 36 for methodsand protocols.

[0358] SAg-encoding nucleic acid is fused in frame (or cotransfected)with nucleic acid encoding a signal transduction protein or HSP.Transfectants are prepared as in Example 1. They are used in vivo as apreventative or therapeutic antitumor vaccine according to Examples 15,16, 18-23. They are also used ex vivo for inducing tumor specificeffector cells for adoptive immunotherapy of cancer (Examples 2-5, 7,15, 16 18-23, 68). The peptide or polypeptide conjugates are also usefulfor the same purposes.

37. SAgs with Specialized Sites for C-terminal GPI Anchoring,Glycosylation, Sulfation, N-Myristoylation, Phosphorylation,Hydroxylation, N-Methylation, Signal Peptide Binding, LPS Binding HSPBinding, Chemokine Binding and Prenylation

[0359] SAg-encoding nucleic acid is fused in frame to nucleic acidsencoding the above “specialized sites” and transfected into tumor cellsor accessory cells The structures of these sites is given in Table 3, p.48 of Rocker R B I et al., J. Nutrition 123: 977-990 (1993).

[0360] Tumor or accessory cells express SAgs in a variety of fashionsafter post-translational modification (Wilkins, M R. et al., ProteomeResearch: New Frontiers in Functional Genomics Springer Berlin, Germany(1997)). For example, myristoylated SAg will bind to surface lipids andwill be minimally secreted. In glycosylated form, the SAg will be routedto the class II pathway and appear bound on the cell surface. When boundto invariant chain, the SAg will be routed to the class II receptor.

[0361] Nucleic acids encoding proteins that active in post-translationalmodification of SAgs are fused in frame to nucleic acid encoding SAgs.These posttranslational modifiable sites include, but are not limitedto, a C-terminal GPI anchor, glycosylation site, palmitoylation site,myristoylation or prenylation site, N-methylation site, hydroxylationsite, phosphorylation site, sulfation site, signal peptidase site,carboxylation site and prenylation sites.

[0362] The incorporation of many membrane proteins into the lipidenvironment is based on sequences of largely hydrophobic amino acidsthat can form membrane spanning domains. However, a large number ofmembrane associated proteins do not display hydrophobic elements intheir primary sequences. The capacity for membrane association in thesecases is often provided by covalent attachment (either cotranslationallyor post translationally) of lipid groups to the polypeptide chain.Acylation of proteins by addition of C14 myristic acid to an N-terminalgly residue or addition of C16 palmitic acid by thioester linkage tocysteine residues is in a variety of positions in SAgs. Palmitoylationof SAgs is not restricted to thioester linkage and may occur throughoxyester linkages to serine and threonine residues. Furthermore,thioester linkage of fatty acyl groups to proteins is not restricted topalmitate. Longer chain fatty acids such as stearic acid (C18) andarachidonic acid (C20) are also produced. The addition of palmitoyland/or myristoyl groups with varying lengths confers additional andsufficient binding energy for hydrophobic binding of proteins toreceptors, membranes or lipid bilayers. The attachment of palmitate issufficient whereas the attachment of myristate is insufficient inisolation. Palmitoylation thus provides a means for membrane anchorageof SAgs and can allow effective concentration of an enzyme or otherregulatory proteins at the membrane.

[0363] Glycosylated SAg is better capable of binding to oligosaccharidereceptors on blood vessels, inflammatory cells or immunocytes. Signalpeptide sequences permit the SAg to be routed to various cell surfacereceptors. Prenylation is important in the membrane attachment andprotein-protein interactions of SAgs and oncogene activation.Prenylation, or post translational enzymatic addition of prenyl,geranyl, farnesyl or geranylgeranyl, involves reactions of a prenyldiphosphate with a cysteinyl sulfhydryl group near the C terminus of theprotein to give a prenyl-S-Cys moiety.; Characteristically theCys-ali-ali-Xaa sequence (“ali” is an aliphatic amino acid; Xaa is anyamino acid) is recognized by the transferase that catalyzes thereaction. When Xaa is serine, alanine or methionine, the protein isfarnesylated; when Xaa is leucine, it is geranylgeranylated.Farnesylation of the protooncogene p21^(ras) is integral both for itsmembrane association and transforming activity. Farnesylated proteinsmediate the induction by IL-1b of NOS whereas a geranylgeranylatedproteins repress this induction.

[0364] Nucleic acids encoding HSPs, along with their promoters, arefused in-frame (or cotransfected) with SAg nucleic acid. These includebut are not limited to two recently discovered HSP genes, orf37 andorf35 in Staphylococcus aureus that are upstream and downstream ofgrpE(hsp20), dnaK(hsp70) and dnaJ(hsp40) in the following sequence:orf37-hsp20-hsp70-hsp40-orf35. The promoters are located upstream oforf37 and upstream of hsp40. These fused proteins are useful aspreventative or therapeutic antitumor vaccines according to Examples15,16, 18-23. They are also useful ex vivo for producing a population ofanti-tumor T cells, NKT cells or NK cells for adoptive immunotherapy ofcancer (Examples 2-5, 7, 15, 16, 18-23).

[0365] Most eukaryotic cells are decorated with chemical groups such asphosphates, methyls, sugars, or lipids during or after their translationfrom mRNA. These extra groups have various functions, often serving asswitches or localization signals. One lipid modification is proteinprenylation in which a 15-carbon farnesyl or 20-carbon geranylgeranylgroup is attached to the protein's —COOH terminus followed by othermodifications (proteolysis, methylation, and palmitoylation).

[0366] Most prenylated proteins are members of signal transductioncascades. For example, the—subunits of heterotrimeric guanosinetriphosphate (GTP)-binding proteins (G proteins) and virtually allmembers of the Ras superfamily of proteins. Farnesylation of H, K, N-Rasis essential for the ability of oncogenic mutants of these proteins totransform cells. 30% of established tumor cell lines containmutationally activated Ras proteins. FTase inhibitors shrink tumors inanimals to an undetectable size with no significant toxicity after weeksor months of exposure. Farnesylation is a prerequisite forpalmitoylation. Palmitoylation of H-Ras occurs only in the plasmamembrane by a putative membrane-bound palmitoyl transferase.Farnesylation may bring a finite amount of H-Ras to all cell membranes,at which point and palmitoylation is required to trap it in the plasmamembrane. H-Ras palmitoylation like G protein-subunit palmitoylation, isreversible and may regulate signal transduction. COOH terminalproteolysis of prenylated proteins and methylation are required forpalmitoylation, membrane binding and Ras function. Prenyl proteinspecific protease and methyltransferase like Ftase may be good targetsfor drugs that prevent oncogenesis.

[0367] Common N terminal additions are fatty acid acylations andglycosylations which provide polypeptide chains with short “lipophilichandles” or recognition sites that serve to facilitate their vectoraltransport or compartmentalization are common N-terminal additions. Forexample, myristic acid in the form of myristyl CoA serves as a substratefor specific N-terminal acylations that are important in anchoringproteins to endoplasmic membranes. The most common C-terminalmodifications are amidations, acylations, polyadenylations and theenzymatic additions of tyrosyl residues. Similarly the C-terminalacylation process is complex. Prenylation occurs at Cys residues isoften associated with proteins that end in Cys-Val-Ile-Ala. The reactionsequence involves (1) a first prenylation (addition of a farnesyl moietyto Cys) followed by (2) cleavage of the Ala, Ile and Val residues and(3) the carboxymethylation of the resulting C-terminal prenylatedcysteine. In addition to providing a membrane anchor, this modificationoften is essential to function of oncogenes such as Ras.

[0368] Two separate and well characterized pathways for carbohydrateaddition: the N-linked dolichol pyrophosphate mediated pathways and theO-linked pathways that utilize UDP sugars as substrates and hydroxylatedamino acid side chains as sites for attachments. Side chains aminophosphorylation of specified proteins usually at tyrosyl or serinylresidues as a way of causing cascade-like amplifications in a metabolicsystem. Methylation and methyl additions can also serve as novel on-offswitches for metabolic processes. The targeted amino acids or methyladditions are lysine, histidine and arginine. In prokaryotes, reversiblemethylations of aspartyl and glutamyl side chains can occur. The bestexample is carboxymethylation of glutamate which is associated withbacterial chemotaxis and is elaborated by the opening and closing ofmembrane ion channels upon methylation and demethylation. Posttranslational modifications can lead to crosslinking and stabilizationof protein matrices. Amino acids such as L-lysine, L-glutamine,L-cysteine and L-tyrosine are utilized extensively as sources forprotein cross-linking. Examples include the extracellular matrix crosslinking of collagen and elastin and the stabilization of keratin-derivedmatrices and tubulin by -glutamyl lysine crosslinks.

[0369] In bacteria, the majority of proteins that form durable wallassociations possess either distinctive N-terminal signals(lipoproteins) or more commonly distinctive C terminal wall associatingsignals although a number of wall associated proteins possess neither ofthese types of signals. A number of wall-associated proteins ingram-positive bacteria are anchored to the external surface of thecytoplasmic membrane via a covalently attached lipid moiety. Bothgram-negative and gram-positive lipoproteins possess similar distinctiveN-terminal signal sequences which contain a tetrapeptide consensus atthe cleavage site consisting of Leu-X—Y-Cys where X and Y arepredominantly small neutral residues and signal and signal peptidasecleavage occurs between Y and Cys. This sequence directs either co- orpost translational modifications involving transfer of glycerol fromphosphatidylglycerol to the +1 Cys, followed by the transfer of fattyacids from phospholipid to the glyceryl-prelipoprotein to produce adiglyceride-prelipoprotein. The C terminal end of a large number of Grampositive wall-associated proteins share common structural features thatare required to localize these proteins in the cells wall. TheseC-terminal structures include a number of distinct features. At theextreme C-terminus there is a stretch of 15-22 hydrophobic residues,followed by a short tail of predominantly charged amino acids.Immediately upstream from this hydrophobic/charged-tail domain, there isa highly conserved Leu-Pro-X-Thr-Gly-X (LPXTGX) motif which is usuallypreceded by a sequence containing a high proportion of regularly spacedprolines. GPI anchors have not been identified on bacterial cell surfaceproteins. But the strong conservation of the LPXTGX motifs and of ahydrophobic/charged tail residue-helical domain are common structuralfeatures that are required to localize these proteins in the cell.

[0370] Protein A is covalently coupled to the cell wall whereas of theproteins are not. Non-covalent interactions may occur in some proteinsholding it in the cell wall while cross-linking occurs around prolinerich region to form peptidoglycans. Hydrogen or water binding sites canbe created by hydroxylation reactions, e.g., hydroxylation of proline incollagen provides sites for intrachain hydrogen and H2O bonding.

[0371] SAg-encoding nucleic acid is transfected into cells together withcoding regions to permit the above post translational modificationswhich contribute to the production of an immunogenic tumor cell,accessory cell (preferably a DC) or a tumor cell/accessory cell hybrid.Such nucleic acids encoding the sites for post-translationalmodifications of SAgs are useful in the structural modification,translocation, cell surface binding and association with keyenergy-producing and signal transduction molecules and receptors. Thecells expressing the products of these post-translational modificationsare useful as a preventative or therapeutic antitumor vaccine accordingto Examples 15, 16, 18-23). They are also useful ex vivo for producing apopulation of tumor specific effector cells (α/β+T cells γ/δ T cells,NKT cells, NK cells) for adoptive immunotherapy of cancer (Examples 2-5,7, 15, 16, 18-23).

38. SAgs and SAg Proteomes for Enhanced Immunogenicity, Specificity andIntracellular Trafficking of Soluble or Cell-Bound Binary or TernaryComplexes

[0372] SAgs with genetically engineered binding sites are provided inorder to enhance their coupling to bioreactive complexes, peptides andLPS's and galactosylceramides. SAgs with a glycosylation otherglycosylceramide binding site bind to glycosylceramide-CD1 orglycosylceramide-CD1 complexes alone in soluble or immobilized form, orcell bound after binding to a receptor on a T cell or NKT cell. SAgs arealso provided with an LPS binding site for binding to soluble,immobilized or cell bound LPS-CD14 complexes.

[0373] SAgs are provided with a glycosphingolipid or glycosylceramidesite by which they can bind to CD1-glycosylceramide orCD1-glycopsphingolipid complexes present in soluble, immobilized form oraffixed to CD1+ cells or NKT cells. Glycosylated SAgs are bound toCD1-glycosylceramide complexes in soluble form or fixed to CD1+ cells orNKT cells. SAgs are also provided with an overexpressed site for MHCclass I molecules, to increase the effectiveness of binding to MHC classI-tumor peptide antigen complexes or TCR-bound MHC class I-tumor peptidecomplexes.

[0374] SAgs are engineered with repeating peptides which bind to the Vβchain to increase clustering. SAgs with an “overexpressed” (in terms ofnumber) SAg receptor site binds to tumor cells expressing SAg receptors.SAgs possess a site for binding HSPs which are useful in immunizingnormal or anergic T cells in a tumor patient. SAgs bind to T cellantagonist MHC-tumor peptide complexes converting the binary complex toa ternary complex with T cell agonist activity. Anergic T cells areactivated by these ternary complexes.

[0375] SAgs are prepared with an overexpressed site for bindingglycosphingolipids or glycosylceramides. These complexes are loaded ontoCD1 receptors of antigen presenting cells and presented to the tumorbearing host either in vivo or ex vivo (Examples 4, 5, 7). SAgs with amyristoylation site will bind to bacterial glycolipids such aslipoarabinan or a mycolic acids The binary complex is then loaded ontoAPCs expressing CD1 receptors. These cells are then used in vivo(Example 14, 15, 16, 18-23) to produce a tumoricidal response.Alternatively, they are used ex vivo to produce tumor specific effectorcells for adoptive immunotherapy (Examples 2, 7, 14, 15, 16, 18-23, 68).

[0376] A SAgs may also be prepared with signal sequences for proteinsorting and intracellular trafficking. Signal sequences comprise shortstretches of amino acids located at the N terminus of a protein, the Cterminus or in the middle of the peptide chain. The physical propertiesof these sequences e.g., their polarity or charge. Signal regions arethree dimensional domains on the surface of a protein made up ofdifferent fragments of the same peptide chain or by different chainsaltogether. Structural signals are recognized and bound by receptorslocated on the membranes of organelles. Signal sequences also serve asrecognition sites for enzymes which modify the proteins altering theirproperties and bring about a change in their fate. Once they havefulfilled their function, some of the signal sequences are removed bysequence specific hydrolases. Signal peptides fused to SAgs guide themto the secretory or exocytosis pathway, or to proteins localized to theendoplasmic reticulum, lysosomes, mitochondria, nucleus, peroxisomes orsecretory vesicles.

[0377] Lipid Rafts Comprising SAg Plus Signal Transduction Molecules

[0378] Lipid rafts are small glycosphingolipid- and cholesterol-richplasma membrane microdomains on the cell surface to which proteins andlipids have variable affinities. They function as platforms for bothsignal transduction and membrane trafficking.

[0379] Sphingolipid-cholesterol rafts are insoluble in the detergentTriton X-100 at 4° C., in which they form glycolipid-enriched complexes(DIGs). Because of their high lipid content, these detergent-insoluble,glycolipid-enriched complexes (DIGs) float to a low density duringgradient centrifugation, which enables any associated proteins to beidentified and distinguishes DIGs from other detergent-insolublecomplexes. Milder detergents such as octylglucoside will solubilizelipid rafts. Glycosphingolipids are insoluble by themselves, andsphingomyelin is resistant to detergent extraction in the presence ofcholesterol.

[0380] A model for the organization of sphingolipid-cholesterol rafts isas follows: Sphingolipids associate laterally with one another, probablythrough weak interactions between the carbohydrate heads of theglycosphingolipids. The sphingolipid head groups occupy larger excludedareas in the plane of the exoplasmic leaflet than do their predominantlysaturated lipid hydrocarbon chains. Any voids between associatingsphingolipids are filled by cholesterol molecules which function asspacers. The close-packed sphingolipid-cholesterol clusters behave asassemblies within the exoplasmic leaflet, where the intervening fluidregions are occupied by unsaturated phosphatidyl-choline molecules.Glycosphingolipids usually carry a long fatty acid which is amide-bondedto the sphingosine base and can interdigitate with the cytoplasmicleaflet of the bilayer. As cholesterol is present in both leaflets, itfunctions as a spacer in the cytoplasmic leaflet as well, filling voidscreated by interdigitating fatty acid chains. Rafts contain proteinsattached to the exoplasmic leaflet of the bilayer by their GPI anchors,proteins bind to the cytoplasmic leaflet by acyl tails (e.g., theSrc-family kinase) or proteins associate through their transmembranedomains, Interactions with raft lipids may be mediated by binding tocholesterol and by acylation of C-terminal cysteines.

[0381] The most important property of the lipid microdomains is thatspecific proteins associate with them. Glycosylphosphatidylinositol(GPI)-anchored proteins and doubly acylated kinases bind to rafts bytheir lipid moieties. Also several transmembrane proteins have beenrecovered in DIGs. Several proteins have been identified as residents ofthese lipid microdomains, including caveolin, GPI-linked membraneproteins, influenza hemagglutinin, the high-affinity IgE receptor, andthe B cell integral membrane protein CD20. In addition, severalmolecules involved in signal transduction have been shown to beassociated with lipid rafts such as the Gα subunits of heterotrimeric Gproteins, the double-acylated Src family protein tyrosine kinases Lck,Lyn, and Fyn, and the Zap70 family protein tyrosine kinase, Syk. Thelipid rafts are also associated with actin and actin-binding proteins.

[0382] The function of rafts in signal transduction is to select andconcentrate molecules or receptors in a micro-environment of themembrane thus speeding up binding during signalling and preventinginappropriate crosstalk between pathways. They are also depots of cargomolecules and transport machinery for trafficking. Clustering of GPIanchors with rafts leads to interaction with signal-transducing proteinsthat then become activated. They facilitate interaction with ligands andeffectors on both sides of the membrane to include kinases and theirsubstrates for signal transduction. Two Src family kinases, Lck and Fyn,both implicated in T-cell activation, are immunoprecipitated withGPI-anchored proteins after Triton X-100 extraction. Indeed, raftsmaintain a pool of the hyperphosphorylated (inactive) Src-like kinaseLck which interacts with rafts by its myristyl and palmitoyl anchor.Trimeric G proteins and Ras also associate with DIGs. Upon cross-linkingthe TCR, its associated signaling molecules and coreceptors acquireincreased affinities toward lipid rafts. Integrity of these domains isrequired for efficient signal transduction by the TCR. Lipids involvedin signal transduction have also been localized to DIGs which includephosphoinositides such as phosphatidyl-inositol-(4,5)-bisphosphate andsphingomyelin.

[0383] Superantigens alone or in combination with other moleculesincluding but not limited to GPI, signal transduction molecules Gproteins, ganglioside/glycolipid transfer agents (See glycolipidtransfer factors-Section 58 and influenza virus hemagglutinin, HA 2 orSendai virus or the heptad repeat unit), and tumor associated antigensare coupled to lipid rafts by methods given in Example 4. The lipidrafts can also contain a α-1-4 digalactosylceramides which are naturalreceptors for SAgs. These derivatized lipid rafts are rapidly integratedinto cell membranes and are used to activate inhibitory or apoptoticpathways in tumor cells. They are useful in transferring receptors totumor cells or T cells which recognize natural ligands for theactivation tumor cell apoptosis. They activate T cells and NKT cells byprovision of superantigens, receptors and key signal transductionmolecules, e.g., lyck or fyn involved in T cell activation. Delivery oftumor associated antigens via lipid rafts, has the added advantage ofstimulating T cells from tumor bearing patients which have beenanergized in the course of tumor growth.

[0384] Lipid rafts are prepared as given in Example 61. They are usefulin vivo as a preventative or therapeutic antitumor vaccine. They areadministered parenterally in doses of 0.01-1 μg in normal saline threetimes weekly either before or after tumor implantation. When given afterimplantation, the treatment is continued for four weeks (Examples 5, 15,16, 18-23, 57-59). They are also useful ex vivo to immunize T or NKTcells to produce a population of tumor specific effector T cells foradoptive immunotherapy of cancer. For ex vivo use the lipid rafts areincubated with T cells (αβT cells, γ/δT cells, NK cells, NKT cells) orantigen presenting cells in doses of 0.1-1 μg for 24 hours after whichthe cells are harvested and administered (Examples 2-5, 15, 16, 18-23,57-59, 68).

[0385] GPI-SAg-Ceramide or GPI-SAg-CD1-Ceramide Complexes Expressed onTumor Cells, Antigen Presenting Cells, Yeast Displays, Sec YeastMutants, APC/tc Hybrid and Shed as Exosomes

[0386] Cells Expressing, Overexpressing or Shedding GPI Proteins arePrepared so that they Comprise Covalently- or Noncovalently Bound Mono-or Diglycosylceramides with Terminal or Subterminal α1-2, α1-4 or α1-6Configurations and SAg Protein or Peptide Moieties.

[0387] The synthetic pathway involves transfection of SAg DNA into atumor cell or accessory cell or a hybrid thereof. The SAg protein istranslated in a precursor form consisting of a receptor-coding regionsandwiched between amino and carboxy-terminal sequence signals. In theendoplasmic reticulum, the signal peptides are cleaved and a GPI anchorcomprising a glycosylceramide optionally bonded to a phytosphingosinechain is attached at a specific site designated w. Furtherpost-translational modifications are made in the Golgi beforetrafficking to the outer leaflet of the plasma membrane. Once GPI-SAgmolecules arrives at the cell surface, they may remain entirely mobilewithin the lipid bilayer or may associate within membrane subdomains.

[0388] GPI-SAgs are released from the cell surface into theextracellular milieu. They leave the cell surface as SAg-glycan-lipidcomplexes, as SAg-glycan complexes or as free SAgs devoid of a GPIanchor. GPI-SAgs released from intact cell are also released free oftheir lipid moiety, hence their designation as LIP(−) GPI-SAgs, whereasthose presumably released with an intact lipid moiety are termed LIP(+)GPI-SAgs. The lipid free moieties are more hydrophilic and thereforesoluble in an aqueous environment, whereas the intactlipid-glycan-protein complexes travel in more hydrophobic environments.In the absence of detergents, the released or “shed” LIP(+)-GPI-SAgs invivo are vesicles with clearly defined lipid bilayers or as hydrophobicaggregates lacking a bilayer morphology. These shed vesicles, oftenreferred to as exosomes, contain many LIP(+) GPI-SAgs. The sheddingprocess itself appears to depend on GPI-proteins, because vesiculationis reduced by 50-90% in cells lacking GPI proteins.

[0389] Shedding is enhanced by treating the tumor cells with 20 mMretinoic acid. In addition high concentrations of glycosphingolipids onthe tumor cell surface are generated by selective transport from thesite of synthesis to the cell surface. Provision of ceramide containingthe α2-hydroxy fatty acid C6OH results in (1) conversion togalactosylceramide, galabiosylceramide and sulfatide and (2) sorting inthe trans-Golgi network to the tumor cell surface. GPI-SAgs remainbiologically active after being released from the outer leaflet of cellmembranes. LIP(+) GPI proteins may also transit to adjacent membraneswhere they associate with the exogenous membranes by incorporatingthemselves into the lipid bilayer in addition to binding to surfacereceptors.

[0390] Additionally, superantigen or oxyLDL receptor nucleic acids aretransfected into yeast sec mutant. The yeast sec mutant,6-4, contains atemperature sensitive mutation in a gene product required for thetransport of secretory vesicles for the trans-Golgi network to theplasma membrane. Gene expression is initiated by an inducible promoterconcomitant results in the arrest of vesicle fusion and the insertion ofSAg or LDL receptor protein in the plasma membrane. Thus gene expressionbegins at the same time that secretory vesicles become unable to fusewith the plasma membrane, ensuring that the desired gene productsaccumulate in the membranes of these vesicles. The purification of thesevesicles is rapid and simple, thereby facilitating the subsequentcharacterization of the desired gene product. Because the Sec6 proteinis known to be involved only in the fusion of these vesicles with theplasma membrane, translocation and processing of proteins in theendoplasmic reticulum and processing in the Golgi are largely unaffectedby the Sec6 mutation.

[0391] The transfected superantigen or LDL nucleic acid (plasmid) isexpressed as superantigen polypeptide or oxyLDL receptor polypeptide invesicles in association with yeast GPI-lipid membrane structures. Thelipid portion of the SAg-GPI-lipid complex comprises a ceramide with aC26 dihydroxy sphingosine or phytosphingosine configuration which isessential for activating NKT cells. The resultingSAg-GPI-phytosphingosine vesicles have the capacity to activate T cellsvia the superantigen and NKT cells via the phytosphingosine and thusproduce a potent anti-tumor effect. Administered preferably by directadministration into the tumor, the oxyLDL receptors induce an excessiveaccumulation of endogenous or exogenously administered oxyLDL and LDL atthe tumor site. The deposited oxyLDL induces apoptosis and foam cellformation in tumor cells and tumor microvascular endothelial cellsresulting in potent tumoricidal response. Optionally,SAg-GPI-phytosphingosine are expressed on these vesicles together withvesicles expressing oxyLDL receptor-GPI-phytosphingosine or oxy LDLreceptors. Tumor associated peptides and polypeptides, tumor apoptosisinducing peptides and polypeptides including but not limited tothrombospondin, angiogenesis inhibitor peptides and polypeptidesincluding but not limited to angiostatin or VEGF are also useful fusedor conjugated to SAgs in the same sec mutant or coadministered with theSAg-sec mutant in a separate sec mutant. Vesicles containing all of theabove constructs including but not limited to SAg-GPI-phytosphingosineor oxyLDL receptor-GPI-phytosphingosine are prepared and isolatedaccording the method of Coury L A et al., Methods in Enzymology 306:169-186 (1999) and as in Examples 4, 5, 7, 42, 50-51.

[0392] All of the constructs given above are administered preferentiallyby direct intratumoral injection as given in Example 20. They are usefulin vivo as a preventative or therapeutic antitumor vaccine according toExamples 2, 7 14-16, 18-23, 36. They are also useful ex vivo to producea population of tumor specific effector cells (αβT, γ/δT, NK, NKT cells)for adoptive, immunotherapy of cancer (Examples 2-5, 7, 15, 16, 18-23,68).

[0393] A yeast cell display system is also used to present SAg to Tcells, NKT cells or NK cells in the context of phytosphingosine groupspresent in the yeast cell membrane. The yeast displays are prepared bythe method of (Cho B K et al., J. Immunologic Methods, 220: 179-188(1998) and used to activate T cells, NK cells and NKT cells. The yeastpresentation has the advantage of presenting as many as 10⁵ proteins percell at the yeast surface potentially allowing multivalent interactionsto occur between the yeast and target cell. The superantigen gene istransfected into the S. cerevisiae. Individual colonies of thetransfected yeast are grown overnight in the Trp-media and harvested inthe log phase. Murine splenocytes (10⁵) are incubated with varyingnumber of yeast cells that bear the superantigen. After 20 h in culture,cells are harvested, washed in PBS and screened for activation markers.In this yeast display system constructs including but not limited toSAg-phytosphingosine or SAg-GPI-phytosphingosine, SAg-lipid orlipoprotein conjugates are presented to T cells and NKT cells to producea population of tumor specific effector cells (T cells, NKT cells or NKcells) useful for the adoptive therapy of cancer under protocols givenin Example 2, 7, 14, 15, 16, 18-23. They are also useful as a vaccine oragainst established tumor as given in Examples 14, 15, 16, 18-23, 68.

39. Effector T Cells: Methods of Lowering Activation Threshold forActivation by SAg

[0394] Tumor peptide MHC complexes are insufficient to activate T or NKTand may even induce antagonism or anergy. SAgs added to the complexesare useful to overcome activating T or NKT cells and overcoming theanergy common in tumor-bearing hosts. To enhance responsiveness totumor-peptide-MHC-SAg complexes and to overcome anergy, it is desirableto reduce the threshold for signal transduction in an effector T or NKTcell population. To accomplish this, nucleic acids encoding SAg-specificTCR Vβ regions are transfected into T or NKT cells to duplicate orotherwise induce overexpression. In addition, measures are taken toalter signal transduction by dimerizing the tyrosine kinase receptors ordeleting the inhibitory region of the TCR.

[0395] Most SAgs show selective binding to well defined segments of theVβ chain of the TCR. The TCR genes are clustered on chromosome 7 andinclude 75-100 V, 2D, 13 J, and 2 Cβ genes. The entire 685-kb humanlocus has been sequenced, the longest contiguous subfamilies thatexhibit >75% sequence identity at the DNA level. The human TCR locus ison chromosome 14 and consists of 42 V genes, 61 J genes and 1 Cβ gene.The TCR chain genes are on chromosome 6 and consist of approximately23V, 2D, 12J, and 2C gene segments. The 2 Cβ genes form clusters withupstream Dβ and Jβ segments: Cβ1 rearranges only with Db1/Jb1 geneswhereas Cβ/Bβ rearranges with both Dβ and Jβ segments. Similarly,functional Vβ genes appear to rearrange to both J clusters in a randomfashion. The β chain transcripts of antigen-specific T cell clonesappear to contain little length variation and harbor conserved Nadditions.

[0396] A mechanism for achieving diversity in variable(antigen-specific) regions of the TCR involves the random addition ofnucleotides inserted at junctional positions during the joining of Vβ DβJ segments. It is at this position that nucleic acids encoding the majorVβ binding site for a specific SAgs are inserted. This overexpressionallows for more selective recognition of SAg and a lower activationthreshold by a SAg that selectively binds at that site.

[0397] Nucleic acid encoding SAg receptor is amplified and transfectedinto T cells to overexpress the SAg receptor on the cell surface These Tcells bind SAgs, and this is linked to appropriate signal transductionpathways that deliver a mitogenic signal to the T cell. One method ofincreasing T or NKT cell reactivity to a SAg is to increase the densityof their SAg receptors. Even in the absence of ligand, the equilibriumis shifted from monomeric inactive receptors to dimeric or oligomericactive receptors. Concomitant expression of the corresponding ligandreinforces the signal. Increased numbers of receptors occur afterincreased transcriptional activation of, or amplification of, the SAgreceptor gene. Amplification is the preferred method.

[0398] The SAg receptor may also be mutated so that it engages inligand-independent dimerization. Examples of such mutations are additionor loss of a cysteine residue in the extracellular domain causingformation of dimeric and disulfide bonded and activated receptors. Inaddition it is possible to dimerize tyrosine kinases by fusing atyrosine kinase catalytic domain to a protein which is a functionaldimer. These fusion partners are able to form homodimers. Such a fusionprotein results in dimerization of kinase domains which allows theirautophosphorylation and activation. Interaction with receptors in amanner which promotes dimerization of two different receptors is anothermethod to enhance receptor reactivity. The kinase domain of a receptormay be mutated to increase catalytic activity or alter substratespecificity. Such mutations expand quantitatively and qualitatively therepertoires of substrates in the target cells and thereby shift thebalance towards activation and transformation. Mutations in regionsinvolved in negative regulation of receptor function also contribute tothe transforming properties. Loss of regions in the C terminus that areregulatory serine phosphorylation or autophosphorylation sites alsocontributes to excessive receptor activity.

[0399] Effector cells as discussed above are prepared as in Examples 4,5, 7. They may also be used in vivo as tumor specific effector (T, NK,NKT) cells for the adoptive immunotherapy of cancer (Examples 2-5, 7,15, 16, 18-23, 68).

40. SAg Nucleic Acids Fused of Cotransfected into Tumor Cell withNucleic Acids Encoding Inducible Nitric Oxide Synthase (iNOS)

[0400] SAg-encoding nucleic acid is fused in frame (or cotransfected)with nucleic acid encoding inducible nitric oxide synthase whichproduces nitric oxide (NO). NO is derived from terminal guanido-nitrogenof L-arginine which is catalyzed by the constitutive or inducible nitricoxide synthase (iNOS). NO is pleiotropic and is a major cytotoxicmediator secreted by activated endothelial cells and macrophages.Production of NO is associated with apoptosis of tumorigenic cells andwith a bystander effect on surrounding non-NO producing tumor cells(bystander effect). Non metastatic tumor cells show high levels of iNOSactivity and NO, whereas metastatic cells do not. There is an inverserelation between production of endogenous NO and the tumor cellssurvivability. In the present invention, tumor cells transfected withSAg-encoding nucleic acid are cotransfected with nucleic acids encodingiNOS. The gene for iNOS has been cloned and characterized by Xie Q etal., Science 256: 225-228 (1992). Tumor cells cotransfected with nucleicacids encoding SAgs and iNOS demonstrate augmented immunogenicity viathe expression of SAg as well as enhanced auto- and bystandertumoricidal capacity via NO production.

[0401] After administration to a patient and colonization of metastaticsites, the transfectants induce a powerful local and systemictumoricidal effect. The presence of NO allows the transfectants to dienaturally via auto-apoptosis within a finite period (usually 72 hours)after administration thus minimizing the risk of inducing activemetastatic disease. These tumor cell transfectants may also be made toexpress oncogenes associated with the metastatic phenotype to promotelocalization of the cells to tumor sites in vivo. The cells may befurther transformed by nucleic acid encoding angiostatin or otherangiogenesis inhibitors for additional tumoricidal potency. Thetransfectant are prepared by methods in Example 1-3 and used as apreventative or therapeutic antitumor vaccine by methods in Example 15,16, 18-23).

41. DCs, Other Accessory Cells and DC/tc Hybrids Expressing and/orSecreting SAg

[0402] Accessory' cells are necessary to generate primary antibodyresponses in culture. Of the various types of accessory cells, DCs arethe most effective APC. DCs are a preferred accessory cell. However, theinvention is not confined to DCs. Any other accessory cell type may beused in place of DCs. In particular, accessory cells are defined inOxford's Dictionary of Biochemistry and Molecular Biology as includingfibroblasts, synoviocytes, macrophages, B cells, Langerhans cells andany other cell type which assists in producing an immune response of anykind.

[0403] DCs have exceptional capability to capture antigens, process andpresent antigenic peptides, migrate to lymphoid organs, and induceprimary immune responses of both CD8+ and CD4+ T cells. The ability ofDCs to act as potent APC in the induction of T cell responses isattributed to the high expression of MHC molecules and adhesion and/orcostimulatory molecules as well as the cells' capacity for to producingcytokines essential for the activation and proliferation of the T cells.

[0404] The number of molecules of antigen-MHC complex on tumor (andinfected) cells is typically small (100 per cell), and are recognized byrare T-cell clones (at a frequency {fraction (1/100,000)}) via a TCRthat has a low affinity (1 M). In vitro or in vivo, only a few DCs arenecessary to provoke a strong T-cell response. In the mixed leukocytereaction, one DC was sufficient to stimulate 100-3,000 T cells. MHCproducts and MHC-peptide complexes are 10-100 times higher on DCs thanon other APCs such as B cells and monocytes. Mature DCs resist thesuppressive effect of IL-10, but synthesize high levels of IL-12 thatenhances both innate (NK cell) and acquired (B and T cell) immunity. DCsalso express many accessory molecules that interact with variousmolecules or receptors on T cells to enhance adhesion and signalling(co-stimulation): examples of such pairs are LFA-3/CD58, ICAM-1/CD54,B7-2/CD86. Tumor cells that express the B7 gene elicit CTLs againstotherwise silent, subdominant tumor antigens. All these properties ofDCs (MHC expression, CD1 expression, secretion of IL-12 and theexpression of co-stimulatory molecules) are upregulated within a day ofexposure to many stresses and “dangers” including microbial products.

[0405] Infected cells and tumors frequently lack the costimulatorymolecules that drive clonal expansion of T cells, the production ofcytokines, and T cell development into killer cells. Located in mosttissues, DCs overcome challenges by capturing and processing antigens,and displaying large amounts of MHC-peptide complexes on their surface.They upregulate their co-stimulatory molecules and migrate to lymphoidorgans, the spleen and draining lymph nodes, where they activateantigen-specific T cells. All of these activities of DCs can be inducedby infectious agents and inflammatory products, so that DCs appear tofunction as “mobile sentinels” that not only bring antigens to T cellsbut also stimulate those T cells in the induction of immunity.

[0406] DCs are present in most tissues in a so-called “immature” state,unable to stimulate T cells. Although these DCs lack the requisiteaccessory signals for T-cell activation, such as CD40, CD54 and CD86,they are well equipped to capture antigens, a key event in the inductionof immunity; the antigen is then able to induce full maturation andmobilization of the DCs. Terminally-differentiated or mature DCs canreadily prime T cells Once activated by DCs, these T cells can completethe immune response by interacting with B cells for antibody formation,macrophages for cytokine release, and target cells resulting in lysis.Thus, immature DCs first handle antigens and then, as mature DCs a dayor more later, they potently stimulate T cells.

[0407] DCs stimulate CTLs, which express the accessory molecule CD8 andinteract with MHC class I bearing cells, to proliferate vigorously. Inthe presence of mature DCs and of IL-12, CD4-expressing T-helper cellsturn into interferon gamma (IFN)-producing TH-1 cells. IFN activates theantimicrobial activities of macrophages and, together with IL-12,promotes the differentiation of T cells into killer cells (CTL). Thecapacity of DCs to produce IL-12 and stimulate TH-1 cells leads tomicrobial resistance. Through IL-4, DCs induce T cells to differentiateinto TH-2 cells which secrete IL-5 and IL-4, activate eosinophils andhelp B cells generate an antibody response, respectively. DCs respond toT cells as well. CD40 and the newly described TRANCE/RANK receptor onDCs are ligated by the TNF (tumor-necrosis factor) family of proteinsexpressed on activated and memory T cells; this leads to increased DCsurvival and, in the case of CD40, upregulation of CD80 and CD86,secretion of IL-2 and release of chemokines such as IL-8 and MIP-1a andb

[0408] Immature DCs capture antigen (and particles and microbes ingeneral) by phagocytosis They then form large pinocytic vesicles inwhich extracellular fluid and solutes are sampled, a process calledmacropinocytosis. Finally, they express receptors that mediateadsorptive endocytosis, including C-type lectin receptors like themacrophage mannose receptor and DEC-205, as well as Fc, located in mosttissues, and Fc receptors. Macropinocytosis and receptor-mediatedantigen uptake make antigen presentation so efficient that picomolar andnanomolar concentrations of antigen suffice, much less than themicromolar levels typically employed by other APCs. However, once the DChas captured an antigen, which also provides signals to mature, itsability to capture antigens rapidly declines, and the cell begins toassemble antigen-MHC class II complexes.

[0409] An antigen enters the endocytic pathway of the DC. DCs producelarge amounts of MHC class II-peptide complexes due to specialized, MHCclass II-rich compartments (MIICs) that abound in immature DCs. MIICsare late-endosomal structures that contain the HLA-DM or II-2M products,which enhance and perform editing functions in the binding of peptide toMHC class II. During maturation of DCs, MIICs convert to non-lysosomalvesicles that discharge their MHC peptide complexes to the surface.

[0410] To generate cytotoxic killer cells, able to eliminate infectedcells, and attack tumor cells and transplanted foreign cells, DCs mustpresent peptides (complexed generally to MHC class I proteins) to CD8+ Tcells. Display of peptide-loaded MHC class I complexes on the DC surfacefollows translocation by a peptide transporter from the cytosol to theER, where complexing occurs and then to the surface.

[0411] Human DCs are characterized by a pattern of surface markers andhave the phenotype CD1a+, CD3^(neg), CD4^(neg), CD8^(neg) CD20^(neg),CD40+ CD86+ in the human. The murine phenotype is and CD3^(neg)CD4^(neg), CD28^(neg), CD8-B220^(neg), CD40+, CD80+ and CD36+.

[0412] Maturation of DCs is required for the initiation of an immuneresponse. Microbial products including whole bacteria and the bacterialcell-wall component LPS and inflammatory mediators such as IL-1, GM-CSFand TNF, stimulate DC maturation, whereas IL10 blocks it. Ceramide,which is induced by maturation signals, shuts down antigen uptake by theDC. Mature DCs express high levels of the NFκB family of transcriptionalcontrol proteins (RelA/p65, RelB, RelC, p50, p52) which regulate theexpression of many gene encoding immune and inflammatory proteins.Signalling through the TNF-receptor family, for example TNF-R(CD-120a/b), CD40, and TRANCE/RANK, results in activation of NFκB.Therefore, to induce an immune response through activation of DCs, apathogen or antigen may have to mobilize the signal transductionpathways of the TNF-R family and TNF-R-associated factors (TRAFs).

[0413] One explanation for the failure of the immune system to eradicatemost immunogenic tumors is the lack of tumor antigen presentation by DCsin vivo.

[0414] Several strategies using tumor antigen-charged DCs as vaccinesfor cancer immunotherapy have been developed. Immunization with DCspulsed with purified tumor-associated peptides or proteins has beenshown to be a powerful method of priming tumor-reactive T cells andinducing host protective and therapeutic antitumor immunity in mice andman. However, such a clinical approach is currently limited due to thepaucity of identified human tumor rejection antigens. The polymorphismof the HLA system has also made it difficult to identifytumor-associated peptides as cancer vaccines. In human melanoma, a classof tumor-associated proteins has been identified. However, it is unclearwhich antigen is the best choice for effective tumor rejection in vivoor how effective any such antigen may be. Thus, immunization withdefined tumor antigens is currently limited to a small number of cancersin which candidate antigens have been identified. Anichini et al, J.Immunol. 156:208-217 (1996), showed that the majority of CTL present inHLA-A2.1+ melanoma patients were not directed to the known tumorantigens, Melan-A/Mart-1, tyrosinase, gp100 or MAGE-3. Therefore,immunization with other, yet unidentified, antigens would be moreeffective in eliciting tumor immunity in these patients. Johnston etal., J. Exp. Med. 183:791-800 (1996) demonstrated that the enhancedimmunogenicity of tumor cells engineered to express the B7-1 gene was aresult of expansion of the antigenic repertoire of the tumor. Thisimplies that vaccination with multiple tumor antigens may be superior touse of a single dominant epitope. Indeed, in situations where atumor-associated antigen remains unidentified, a novel approach isneeded for presentation of that antigen by a professional APC.

[0415] An alternative approach, not encumbered by these limitations, isto use unfractionated tumor peptides or tumor proteins as a source oftumor antigens. Two studies have shown that administration to mice ofAPC (from the spleen) or epidermal Langerhans cells pulsed with tumorfragments resulted in protective immunity against tumor challenge.Zitvogel et al., J. Exp. Med. 183:87-97 (1996) showed that vaccinationof mice with bone marrow-derived DC pulsed with unfractionated tumorpeptides reduced the growth of subcutaneously established, weaklyimmunogenic tumors. Thus, immunization with multiple tumor antigens maybe superior to use of a single dominant epitope.

[0416] One approach to overcome the possible drawbacks of unfractionatedtumor antigens is to use mRNA from tumor cells as a “source” of antigen.mRNA can be amplified from a very small number of cells, permitting thegeneration of sufficient amounts of antigen from minute amounts of tumortissue Moreover, tumor-specific mRNA can be enriched by subtractivehybridization to remove RNA that is common to normal tissue. Thisincreases the levels of the relevant tumor-specific antigen(s) that canbe achieved, and hence, the potency of the vaccine. More importantly,this approach reduces the concentration of nonspecific antigens or,possibly, self-antigens, thereby lessening the potential forautoimmunity. Pulsing DCs with RNA is known to be effective inempowering them to induce CTL responses and tumor immunity.

[0417] The fusion of tumor cells with DCs is another approach togenerate a hybrid vaccine that has both potent antigenprocessing/presenting power along with the endogenous expression ofmultiple tumor antigens. Such a hybrid cell would be more effective ininducing antitumor immunity. Gong et al., Proc. Natl. Acad. Sci USA26:6279-6283 (1998), demonstrated that fusion of a relativelyimmunogenic mouse tumor, MC38 carcinoma, with syngeneic DCs resulted ina vaccine that induced (1) T cell protective immunity against tumorchallenge and (2) rejection of an established tumor. Wang et al., J.Immunol. 161:5516-5524 (1998) used the poorly immunogenic B16 (B16.F10)melanoma which does not express MHC and costimulatory molecules.Immunization with irradiated B16 tumor cells failed to induce systemicimmunity or elicit functional tumor-reactive T cells. RMA-S is aRauscher MuLV-induced T cell lymphoma originating in a C57BL/6 (“B6”)mouse that is genetically defective in TAP, and thus, does not processendogenous antigens for binding to MHC. Fusion of DCs with syngeneictumor cells generated hybrid cells that expressed both DC-associatedaccessory molecules important for antigen presentation and tumor-derivedantigens. The DC/tc hybrids were processed and presentedtumor-associated antigens and elicited tumor-reactive CTLs. Vaccinationof B6 mice with B16/DC hybrid cells induced partial protective immunityagainst tumor challenge. Immunization with B16/DC or RMA-S/DC hybridcell vaccines primed lymph node (LN) T cells, which, after expansion exvivo, were active in adoptive immunotherapy. The transfer of suchvaccine-primed, expanded T cells into tumor-bearing mice reduced thenumber of established B16 pulmonary metastases and, in the case ofRMA-S/DC, effectively eradicated disseminated FBL-3 tumor.

[0418] The present invention includes a hybrid cell made from fusion ofa tumor cell and a DC cell further transformed or transfected with aSAg. Nucleic acids encoding SAgs may be introduced into either the tumorcells or the DCs prior to fusion as in Example 1, 2, 3, 25, 26. Thisfused cells are prepared as in Example 24, 25 and their phenotypeestablished by the retention of DC characteristics, tumor cell antigensand the expression of SAg (Example 25). By virtue of these multiplefeatures, this SAg-expressing DC/tc has the unique capacity activatemaximally an anti-tumor immune response.

[0419] SAg stimulation is known to activate CD4+ and CD8+ T cells torecognize and lyse tumors specifically both in vitro and in vivo. The DCcomponent of the hybrid cell provides optimal tumor antigen presentationdue to its enormous surface area together with natural expression ofcostimulatory molecules B7.1, B7,2, adhesion molecules ICAM-1 andICAM-3, MHC class I and class II and CD1 receptors. B7.1, in particular,provides a basis for expanding the epitope recognition spectrum fromdominant to subdominant epitopes. The expressed SAg confers upon thehybrid cell an augmented capacity to activate various classes of cellsthat mediate both innate and “acquired” or adaptive immunity, includingCD4+ and CD8+ T cells, NK cells and NKT. The SAg also contributes togeneration of TH-1 cytokines by this class of T helper cells whichcontributes to an optimal anti-tumor response. The DC/tc hybrid thatexpresses and/or secretes SAg is abbreviated herein as an “S/D/t” celland combines the potent activating properties of SAg with thespecialized (tumor) antigen presenting capacity of the DC and the tumorantigens provided endogenously by the tumor cell partner. This S/D/tcell thus consolidates in a single cell the capacity to unleash andamplify the full weight of the host immune response specifically againsta selected array of tumor associated antigens.

[0420] The present invention also includes the additional introduction,into the S/D/t cell of with additional nucleic acids. In one embodiment,the additional nucleic acid encodes the particular galactosyltransferaseenzyme that catalyze the synthesis of the “heterograft epitope” Gal. Inanother embodiment, the additional nucleic acid encodes enzymes thatsynthesize galactosylceramide which is the “natural” epitope recognizedby the invariant chain of NKT cells.

[0421] To summarize the foregoing section, the present inventionincludes DCs, other accessory cells or hybrid DC/tc, each transformed toexpress SAgs as described in Examples 1 and 3. The transformed (ortransfected) hybrid cell, the S/D/t cell, expresses (1) the majoraccessory molecules of DCs cells (such as CD40, CD80 and CD86, MHC classI and II and CD1); (2) tumor associated epitopes provided by the tumorcell fusion partner; and (3) SAg either membrane bound, secreted or bothwhich activates T cells, NK cells and NKT cells to produce a specific orselective tumoricidal response.

[0422] While the tumor S/D/t cells are preferred, SAg-transfected DCs orother accessory cells are also effective in inducing antitumorresponses. These are used as a preventative or therapeutic antitumorvaccine, or ex vivo to stimulate a population of T cells, NK cells orNKT cells for adoptive therapy of cancer (Examples 29).

42. DCs Expressing SAg and Tumor Associated Antigens—Production byProcessing of Apoptotic Tumor Cells or Tumor Cell Lysates

[0423] DCs expressing SAg and tumor associated antigens are preparedwithout cell fusion (Example 28). Apoptotic, SAg transfected tumor cellsare prepared by first transfecting tumor cells with SAg (Example 1) andthen inducing apoptosis by irradiation or other methods well known inthe art (Example 28). DCs express α_(v)β₅-binding integrins and secretethrombospondin which ligates vitronectin expressed on the surface of theapoptotic tumor cell. DC surface CD36 binds to its natural ligand,sequestrin, also expressed on apoptotic tumor cells. The apoptoticSAg-expressing tumor cells are phagocytosed and processed by DCs underconditions described in Example 28.

[0424] In another embodiment, lysates of tumor cells optionallyexpressing SAg are also used as above. Tumor cells are first transformedto express SAg and then lysed (Example 28. These lysates are “fed” topDCs as in Example 28. DCs treated in this way can now presenttumor-associated antigens along with SAg to the immune system.Alternatively, DCs are first transformed to express SAg, and these cellsare allowed to phagocytose or process apoptotic tumor cells or lysates.Optionally the tumor cells may have been previously genetically modifiedwith nucleic acids so that they synthesize β1,3-glucan, LPS,peptidoglycan or Gal Cer.

[0425] The resulting SAg-expressing DC, after phagocytosing apoptotictumor cells or Iysates, expresses MHC class II, costimulatory moleculesCD40, CD80 and CD86, together with SAg and tumor associated antigen. Theadditional expression of SAg in this system permits more potentactivation of T cells, NKT cells and NK cells which recognize the tumorassociated antigens expressed on the DC surface in the context of MHCand costimulatory molecules.

[0426] In another embodiment, tumor cells are fused to mammalian cellsincluding but not limited to proximal tubular epithelial cells, otherkidney cells including the Madin-Darby canine kidney (MDCK) cell linewhich express an abundance of alpha anomeric digalactosylceramides whichare natural superantigen receptors. In an additional embodiment, tumorcells are fused to cells including but not limited to amphibianintestinal cells which express a high level of phytosphingosine (seeExample 25 for cell fusion methodology). The resulting hybrid cellsexpress tumor associated antigens and either galactosylceramides orphytosphingosine. Alternatively, exogenous galactosylceramide orphytosphingosine from the cell membranes of the above kidney oramphibian cells are incorporated into intact tumor cells by methodsgiven in Section 38 and Example 5. These hybrid tumor cells or tumorcells with newly acquired membrane glyco- or phytolipids (TCGP) arefurther transfected with superantigen nucleic acids to produce hybridtumor cells or TCGP expressing superantigens, tumor associated antigens,galactosylceramides and/or phytosphingosine (Example 1). These hybridtumor cells or TCGP are potent activators of T cell, NK cell and NKTcells

[0427] The DCs, hybrid tumor cells, or TCGP given above are used in apreventative or therapeutic antitumor vaccine (Example 29) or ex vivo toactivate tumor specific effector cells, T cells, γ/δT cells, NKT cells,NK cells for the adoptive immunotherapy (Example 29).

43. DCs Expressing or Secreting SAg Cotransfected with a TumorAssociated Antigen or “String of Beads” Tumor Antigens

[0428] When a dominant tumor associated antigen (protein) is known,nucleic acid encoding such an antigen are used to transform DCs whichalready express or secrete SAg (Example 35). Antigens identified by“SELEX” technology which consists of nucleic acids encoding tumorantigens from distinct structural and functional categories of humantumor associated antigens, including mutants, differentiation variants,splice variants, amplified/overexpressed antigens or retroviral antigensmay be used. Nucleic acids encoding tumor antigens used to transfectSAg-expressing DCs or DC/tc hybrids. This invention contemplatestransfecting with individual nucleic acids encoding a single antigen, ormultiples as in a “string of beads” carried by adenoviral or othervectors known in the art (Example 35). Nucleic acids encoding a “stringof beads” or tumor associated antigens identified by SELEX may be fusedin frame (or cotransfected with) SAg-encoding nucleic acid into DCs orDC/tc. These SAg- and tumor antigen-expressing DCs or DC/tc hybrids areused as a preventative or therapeutic antitumor vaccines (Example 29) oras stimulators ex vivo of T cells, NKT cells or NK cells for adoptiveimmunotherapy (Example 29).

[0429] Furthermore, nucleic acids encoding proteins listed in Tables I,II, IV and V, for example, angiostatin, protein A, erb/Neu and HSPs,staphylococcal collagen adhesin, are introduced into and expressed intumor cells or DCs that express or secrete SAg, or into S/D/t cells.These cells that coexpressing the proteins and peptides of Tables I, II,IV and V together with SAg are useful as preventative or therapeuticantitumor vaccines (Example 29) or as stimulators ex vivo that activatetumor specific effector cells (T cells, γ/δT cells, NKT cells, NK cells)for adoptive immunotherapy (Example 29, 68).

44. Naked DNA or RNA Obtained from the Various Cells Described Abovethat Express and/or Secrete SAg

[0430] DNA containing the CpG backbone is extracted from tumor cells orDCs that express/secrete SAgs or S/D/t cells (Example 30-34). Thepreferred source of DNA or RNA is the S/D/t cells DCs or tumor cellsexpressing SAg are also useful. Alternatively, the DNA or RNA can beobtained from DCs, tumor cells or DC/tc into which SAgs were introducedby the cells having phagocytosed SAg-transformed apoptotic tumor cellsor tumor cell lysates.

[0431] The extracted DNA or RNA is used as a naked DNA or RNApreventative or therapeutic vaccine (Examples 30-34). Alternatively,this nucleic acid material may be used ex vivo to activate T cell, NKTcells or NK cells adoptive immunotherapy (Example 1, 31, 33). Thisextracted DNA or RNA may be used in an initial step of inducing immunereactivity in regional lymph nodes of tumor bearing subjects. After this“priming,” T cells, NKT cells and/or NK cells are harvested from theselymph nodes, expanded in culture in the presence of additional SAg,SAg-expressing DC or tumor cells, or S/D/t cells to generate a T cell,NKT cell or NK cell population for adoptive immunotherapy (Examples 29).

[0432] DNA or RNA for immunization may also be obtained from the variouscells described above that express SAg, and which additionally expressor several Staphylococcal adhesins, β-glucans, LPS, peptidoglycans,teichoic acids, mannose, mannan, protein A and/or their respectivebinding proteins.

[0433] Also useful for naked nucleic acid immunization are bacterial orinsect nucleic acids (with CpG motifs) which encode enzymes thatcatalyze the biosynthesis of β-1,3-glucans, LPS, peptidoglycan, α-gal,GalCer, teichoic acids, mannan or mannose. Also useful are bacterial orinsect nucleic acids that encode the binding proteins for the abovecarbohydrate-based molecules, glycoprotein lectins that bind thecarbohydrate structures, or protein A. Such nucleic acids are used toco-immunize along with SAg expressing DCs or tumor cells or S/D/t cells.Such combined vaccine preparations are used as a preventative ortherapeutic antitumor vaccines (Examples 29, 30). Alternatively, theymay be used to initiate adoptive T cell therapy by priming regionallymph nodes T cells which are harvested, expanded in vitro bystimulation with S/D/t cells, accompanied by, or followed with IL-2. Thetumor antigen-sensitized T cells are reinfused into subjects asdescribed in Example 29.

45. Exosomes Derived from (1) SAg-Expressing Tumor Cells (2) SAgExpressing-DCs (3) S/D/t Cells or (4) DC/tc Hybrid Cells

[0434] MHC-peptide complexes accumulate in endosomes and lysosomes,which compartments contain MHC class II-enriched internal vesicles thatare released outside the cell following direct fusion of the externalendosomal membrane with the plasma membrane. These vesicles, termed“exosomes” are capable of stimulating CD4+ T cell clones in vitro. Inaddition, tumor peptide-pulsed DC-derived exosomes prime specific CTLsin vivo leading to a T cell-dependent eradication or suppressed growthof established murine tumors. In the present invention, the exosomeswhich have SAgs in addition to tumor associated antigens and MHC class Iand class II molecules are prepared. Such preparations are significantlymore potent in their ability to induce shrinkage of established tumorsand prevent tumor outgrowth.

[0435] Exosomes are prepared from (1) tumor cells or DCs which have beentransfected with SAgs (2) S/D/t cells, (3) DCs or hybrid DC/tc whichhave phagocytosed SAg-expressing apoptotic tumor cells or tumor celllysates (Example 36). In the above hybrids, either the DC or tumor cellmay be transfacted with SAg encoding nucleic acid prior to fusion. Theresulting exosomes express MHC class I and class II molecules, SAgs andtumor associated antigen. In order to ensure the routing of thetransforming SAg to exosomes, the SAg-encoding nucleic acid shouldinclude a sorting signal to localize the SAg to the exosome. These cellsmay be pulsed with tumor associated antigens shortly before isolation oftheir exosomes. The isolated exosomes are used as preventative ortherapeutic antitumor vaccines (Example 36) or as stimulators ex vivothat activate tumor specific effector cells (T cells, γ/δT cells, NKTcells, NK cells) for adoptive immunotherapy (Example 36). These variousexosome preparations are extremely effective inducers of anti-tumorresponses.

46. Cell Surface Display of Recombinant SAg and Tumor AssociatedAntigens in Bacteria

[0436] Heterologous proteins and various carbohydrate-containingmoieties, displayed on the surface of bacterial cells often act as majorantigenic systems that stimulate anti-tumor immunity. Such antigensinclude GalCer, α-gal, β1,3-glucans, LPS, peptidoglycans, teichoic acidsand mannan. These structures will be referred to below collectively as“anti-tumor motifs.” These structures are created by the action ofenzymes encoded by a number of bacterial and fungal genes. For example,Sphingomonas paucimobilis expresses GalCer, or Klebsiella aerobacterexpresses -Gal and LPS, and Cryptococcus expresses β1,3-glucan. Becausenot all the genes responsible for the biosynthesis of these moleculeshave not been identified, it is difficult to isolate them and introducethem into mammalian cells. These structures are, however, biosynthesizedin abundance by bacteria. Immunization with live recombinant bacteriainduces both local and systemic immune responses suggesting thatgram-positive bacteria might constitute potential live bacterial vaccinedelivery systems. The surface molecules of gram-positive bacteria seemto be more permissive for the insertion of extended sequences of foreignproteins than are gram-negative bacteria, in which both translocationthrough the cytoplasmic membrane and correct integration into the outermembrane are required for proper surface exposure.

[0437] In the present invention, different bacterial surface displaysystems are used to express natural anti-tumor motifs for developinglive bacterial vaccine vehicles. SAgs are provided to bacteria which donot naturally biosynthesize them so that they are expressed togetherwith natural anti-tumor motifs made in the bacteria. These bacteria arethen used as preventive or therapeutic antitumor vaccines (Example 28).

[0438]Sphingomonas paucimobilis bacteria express GalCer which canactivate the V14 invariant chain expressed by NKT cells. These cellsrecognizes the galactosylceramide epitope. NK cells, using their NKP1-1receptors, recognize carbohydrate units such as 1b,3-glucans expressedwidely on fungi. NK cells are activated directly by SAgs. Furtherproliferation is induced by interferon produced by T cells in responseto the SAg. Humans have natural antibodies specific for the αGalepitope. This epitope is constitutively expressed on several bacteriaincluding Klebsiella aerobacter and E. coli.

[0439] Coexpression of SAg with the above anti-tumor motifs inrecombinant bacteria or fungi provides potent signals to activate NKTcells, T cells and NK cells and to induce production of TH-1 cytokines.The adhesion molecule VCAM-1 expressed by some SAgs such as enterotoxinC contributes to the process by costimulation. Therefore, the SAgexpressing bacteria (whether natural or transformed) are capable ofactivating all of the major cell types involved in the anti-tumorresponse.

[0440] In the present approach, the preferred SAg is SEB. SEB isintroduced for surface display into S. carnosus. E. coli-staphylococcusshuttle vectors are constructed by taking advantage of (1) the promotersignal sequence and propeptide region from the lipase gene constructderived from S. hyicus and (2) the cell surface attachment part ofstaphylococcal protein A. A 198-amino-acid region, designated ABP(albumin binding protein), is expressed adjacent to the cell wall toincrease accessibility to the surface-displayed target peptides.Staphylococcal enterotoxin B is introduced between the lipase propeptideand the ABP region and the surface exposure of the three differentregions are tested separately with different assays.

[0441] These recombinant bacteria are useful as a preventative ortherapeutic antitumor vaccine (Example 28) or as stimulators ex vivo oftumor specific effector cells (T cells, γ/δT cells, NKT cells, NK cells)for adoptive immunotherapy (Example 28, 68).

47. Introduction of Staphylococcal Collagen Binding Adhesins into DCs,Tumor Cells or S/D/t Cells

[0442] Nucleic acids encoding SAgs are transfected into these variouscells, as described above, together with nucleic acids encodingStaphylococcal collagen adhesin. Mice immunized with a recombinantfragment of the collagen adhesin were protected against Staphylococcusaureus-mediated septic death. Sera from S. aureus-immunized micepromoted phagocytic uptake (opsonized) and enhanced intracellularkilling of the bacteria compared to sera from control mice.

[0443] The collagen binding adhesin is isolated from S. aureus strainCowan. Sequencing of the cloned corresponding gene cna revealed a133-kDa polypeptide (close to that of 135 kDa reported for the nativeprotein). This protein is proposed to consist of a signal sequence (S)followed by a large nonrepetitive region (A). Immediately following theA region are three consecutive repeats of a 167 amino acid long unit(B1, B2, B3). A cell wall (W) region consisting of 64 amino acidproline-and lysine-rich domain is followed by stretch of hydrophobicamino acids (M), presumably constituting the cell membrane spanningregion. Finally, the C-terminus (C) is made up of a few positivelycharged amino acids. This model structure is used as the starting pointto identify the collagen binding domain. The ligand binding site islocalized within the 135-kDa S. aureus collagen adhesin. The collagenbinding domain is localized to a 168 amino acid long segment [CBD(151-318)] within the N-terminal portion of the adhesin.

[0444] Using biospecific interaction analysis, bovine collagen was foundto contain eight binding sites for CBD (151-318), two of which were highaffinity and six low affinity. The deduced amino acid sequence of theligand binding domain of the collagen adhesin is presented. Subsequentlya discrete collagen-binding domain within the collagen adhesin wasidentified and localized to a region between amino acids Asp209 andTyr233. The FDA strain 574 of S. aureus encodes a 1185 amino acidcollagen adhesin. The complete nucleotide sequence of the cna gene aswell as a schematic model of the collagen adhesin have been published.The overall structure resembles that of other gram positive surfacestructures. The lysine and proline rich hydrophilic region which followsthe repeated domains resembles a structure in protein A, staphylococcalfibronectin receptor and streptococcal protein G and M proteins. Alsopresent is the hexapeptide LPKTGM which is similar to the consensussequence LPXTGE which is conserved among other gram positive surfaceproteins. The hydrophilic region is thought to mediate the binding ofthe protein to the cell wall. The presence of hydrophobic amino acidswhich may traverse the membrane followed by a C-terminal cluster ofpositively charged residues, possibly located on the cytoplasmic side ofthe membrane, is characteristic of staphylococcal cell surface proteins.

[0445] In the collagen adhesin, a 29 amino acid signal peptide at theN-terminus is followed by a large nonrepetitive A domain, and the highlyhomologous domains B1, B2 and B3 (probably a result of a series ofstepwise gene duplication events). Collagen binding receptors have beenfound on other species of bacteria such as the 75× adhesin ofuropathogenic E. coli. Type 3 fimbrias from pathogenic enteric bacteria,some species of oral streptococci, Streptococcus pyogenes, Yersinia andTreponema pallidium have all been reported to bind various forms ofcollagen. Thus the collagen binding appears to be a common modality usedby pathogenic bacteria of a diverse group to adhere selectively to hosttissues and form a focus of infection.

[0446] Nucleic acids encoding staphylococcus collagen adhesin areintroduced into SAg-expressing tumor cells or DCs, or S/D/t cells. Thecells co-expressing the staphylococcal collagen adhesin with SAgs areuseful as a preventative or therapeutic antitumor vaccines (Example 28)or as stimulators ex vivo that activate tumor specific effector cells (Tcells, γ/δT cells, NKT cells or NK cells) for adoptive immunotherapy(Example 28, 68).

48. Co-Expression of Anti-Tumor Motifs or their Binding Proteins withSAg

[0447] Tumor cells or DCs expressing SAgs, or S/D/t cells, aretransformed with nucleic acids encoding enzymes that catalyze thebiosynthesis of anti-tumor motifs, including the α-gal epitope, theGalCer epitope, β-1,3-glucans, LPS, peptidoglycan, teichoic acids or aprotein or peptide such as Staphylococcal adhesins, protein A, and/orthe binding proteins for the above motifs or proteins. Transformationmay be achieve using bacterial plasmids or nucleic acids integrated intoan appropriate viral vector. These antigenic structures are fundamentalunits recognized in the primitive host defense mechanisms (“innateimmunity”) of invertebrates, but also evoke responses in mammalianimmune systems via the TOLL and NFκB systems.

[0448] DNA encoding the galactosyltransferase that synthesizes thesaccharide structure containing the αGal epitope, and gene clustersencoding the biosynthetic pathway for LPS are described in Schnaitman CA, et al., Microbiol. Rev. 57: 655-682 (1993). DNA is extracted frombacteria which biosynthesize these molecules and used to transfect DCs,tumor cells, or S/D/t cells For creation of the GalCer structure, thesource of DNA is Sphingomonas paucimobilis organisms. Nucleic acidsencoding the pathways for biosynthesis of β-1,3-glucans, peptidoglycans,and protein A have been cloned from insects and Staphylococcus aureus,respectively. These nucleic acids are cloned into suitable expressionvectors and introduced into the target cells. Resulting S/D/t cells thusexpress SAg as well as the anti-tumor motif structure.

[0449] S/D/t cells that co-express Gal can interact with and stimulateNKT cells through the Va14 invariant chain which naturally recognizesthe -galactosylceramide epitope. NK cells, via their NKP1-1 receptors,will recognize carbohydrate units such as β-1,3-glucans on the S/D/tcells. The co-expressed SAg induces further NKT cell expansion. The SAgis also capable of inducing massive proliferation of conventional Tcells which can be further promoted by the co-expression of B7-1, B7-2and ICAM-1 which are normally expressed on DCs. VCAM-1, expressed bysome SAgs such as enterotoxin C, also is capable of contributing to thisstimulation. As indicated above, NK cells are activated directly orindirectly by T-cell derived interferon.

[0450] The S/D/t cells (as well as tumor cells or DCs expressing SAg)that also express one or more of the anti-tumor motifs are capable ofactivating all of the major cell types involved in anti-tumor immunity:T cells specific for peptides, NKT cells reactive with lipoproteins andglycosylceramides and NK cells that recognize for oligosaccharides.These cells are useful as preventative or therapeutic antitumor vaccines(Examples 29) or as stimulators ex vivo that activate tumor specificeffector cells (T cells, γ/δT cells, NKT cells or NK cells) for adoptiveimmunotherapy (Example 29).

49. SAgs Combined with Low Density Lipoproteins (LDL), Oxidized LDL (oxyLDL) Oxidized LDL Mimics and Apolipoproteins

[0451] In the present invention, low density lipoproteins (collectivelyLDL) intermediate density LDL (IDL), chylomicrons, very low densitylipoproteins (VLDL), oxidized LDL (oxyLDL), oxyLDL mimics as well as andapolipoproteins including but not limited to apolipoprotein (a), B100and E4 are conjugated to superantigens and are useful as anti-canceragents alone.

[0452] LDLs, oxyLDLs and apolipoproteins are physically trapped or bindto receptors expressed in the dense network of randomly branching bloodvessels and sinusoids of the tumor neovasculature and have the capacityto deposit or bind to LDL receptors on the tumor endothelium and toscavenger receptors on macrophages OxyLDL or apolipoproteins bound totumor endothelium or macrophages they induce apoptosis or they promoteinflammation by activating vascular cells and macrophages to generatecytokines, chemoattractants and tissue factor.

[0453] Superantigens in nucleic acid or polypeptide form are conjugatedto the lipoproteins and amplify the inflammatory effect of thelipoproteins by inducing apoptosis of endothelial cells, upregulatingendothelial cell integrins, adhesins and procoagulant activity whileactivating macrophages and immunocytes. Any tumor which isneovascularized is eligible for this therapy. These conjugates thereforehave the advantages of localizing to disseminated and neovascularizedtumor, inducing apoptosis and initiating a powerful anti-tumor response.

[0454] Lipoproteins

[0455] Lipoproteins are globular particles of high molecular weight thattransport nonpolar lipids (primarily triglycerides and cholesterolesters) through the plasma. Lipoproteins have been classified on thebasis of their densities into five major classes: chylomicrons, very lowdensity lipoproteins (VLDL), intermediate-density lipoproteins (IDL),low-density lipoproteins (LDL), and high-density lipoproteins (HDL). Thephysical-chemical characteristics of the major lipoprotein classes arepresented in Table The core of the spherical lipoprotein particle iscomposed of two nonpolar lipids hydrophobic lipids, triglyceride andcholesteryl ester, which are present in different lipoproteins invarying amounts. This hydrophobic core accounts for most of the mass ofthe particle, and consists of triglycerides and cholesterol esters invarying proportions. Surrounding the core is a polar surface coat ofphospholipids that stabilize the lipoprotein particle so that it canremain in solution in the plasma. Variable amounts of unesterifiedcholesterol are interdigitated with the phospholipids of the surfacecoat. In addition to phospholipid, the polar coat contains small amountsof unesterified cholesterol. Each lipoprotein particle also containsspecific proteins (termed apoproteins) that are exposed at the surfaceand extend into the core. The apoproteins bind specific enzymes orreceptors on tumor microvascular cells.

[0456] Chylomicrons

[0457] Chylomicrons are large lipoprotein particles formed withinintestinal epithelial cells from dietary triglycerides and cholesterolwhich are secreted into the intestinal lymph and pass into the generalcirculation where they adhere to LDL receptors on the tumormicrocapillaries. Chylomicron remnants are removed by both LDL receptorsand LDL-receptor related protein/alpha2-macroglobulin receptor (LRP).While bound to these endothelial surfaces, the chylomicrons are exposedto the enzyme lipoprotein lipase. The chylomicrons contain anapoprotein, apoprotein CII, that activates the lipase, liberating freefatty acids and monoglycerides.

[0458] Very Low Density Lipoprotein (VLDL)

[0459] Very low density lipoprotein (VLDL)are triglyceride richparticles which are secreted from the liver into the bloodstream afterconversion of carbohydrate to glycerol-esterified fatty acids to formtriglycerides. VLDL particles are relatively large, carry 5 to 10 timesmore triglycerides than cholesteryl esters, and contain a form ofapoprotein B, designated B 100, that differs from the apoprotein B48 ofchylomicrons. The VLDL particles are transported to LDL receptors ontumor microcapillaries, where they interact with the same lipoproteinlipase enzyme that catabolizes chylomicrons. VLDL also binds to the VLDLreceptor via apolipoprotein E and lipoprotein lipase. Bothapolipoprotein E and lipoprotein lipase are constituents of chylomicronremnants which are a physiological ligand for the VLDL receptor.

[0460] Plasma Apolipoproteins

[0461] Plasma apolipoproteins have a central role in plasma lipidtransport. Central to the functions of all apolipoproteins (apo) isspecialized regions termed amphipathic helices which have the ability tobind phospholipids. The amphipathic helices in apoA-I, apoA-II, andapoC-III comprise multiple repeats of 22 amino acids or 22-merperiodicity each consisting of a tandem array of two 11-mers which tendto begin or end with a proline. The characteristic spatial arrangementof the hydrophobic and hydrophilic amino acids within the amphipathichelices is that the hydrophobic face is intercalated between the fattyacyl chains of phospholipids and the hydrophilic face is located closeto the polar head groups of phospholipids. Such an orientation allowsthe interaction of protein domains with lipoprotein-modifying enzymesand cellular receptors that control the catabolism of lipoproteins (Lp)and their removal from the circulation. The major apolipoproteins usefulin the present superantigen-apolipoprotein conjugates are as follows:

[0462] Apolipoprotein (a)

[0463] Apolipoprotein (a) (Lp (a)) is made by hepatocytes and issecreted into plasma where it forms a covalent linkage by a singleinterchain disulfide bond to a unique multikringle glycoprotein, withapo B 100 of LDL to form lipoprotein(a). called apolipoprotein(a).Protein apo (a) has structural similarities to plasminogen and consistsof multiple bent repeats of amino acid sequences. Apolipoprotein (a)exists in polymorphs distinguished by molecular weights. The molecularbasis for the size variation of apo[a] is primarily due to multipleapo[a] alleles that differ in the number of kringle type 2 (plasminogenkringle type 4) repeats. Minor variability in apo(a) size might be dueto differences in glycosylation, as carbohydrates make up 25-40% of theapo (a) weight.

[0464] A close structural similarity exists between apo(a) andplasminogen a protease zymogen whose active form cleaves fibrin todissolve blood clots, is activated by tissue and urokinase plasminogenactivators via cleavage at a specific arginine residue. Indeed, in-vitroand ex-vivo studies have shown that apo(a) binds to immobilized fibrin(fibrinogen), to the plasminogen receptor on endothelial cells andcompetes with tissue plasminogen activator in converting plasminogen toplasmin. Lipoprotein(a) also competes with plasminogen for itshigh-affinity binding sites in endothelium, platelets, and macrophages.Because of structural homology with plasminogen apo(a)I competitivelyinhibits fibrin-dependent activation of plasminogen to plasmin andplasmin-mediated activation of cytokine transforming growth factor-b.Hence, Lp(a) is capable of interfering with the fibrinolytic process byacting as a procoagulant. The colocalization of apo(a) with fibrin(fibrinogen) in the arterial wall further suggests that Lp(a) isthrombogenic.

[0465] Lp(a) is a poor ligand for the LDL receptor and is consequentlytaken up and degraded by unregulated mechanisms, leading to tissueaccumulation. Lp(a) is targeted to uptake by macrophages, presumablythrough the scavenger-receptor pathway. Owing to the lower β-carotenecontent, Lp(a) may be more easily oxidized than LDL. Oxidized Lp(a) suchas Lp(a) modified by malondialdehyde, a product generated in vivo fromaggregated platelets, is avidly taken up by monocyte-macrophages.through the scavenger-receptor pathway. Lp(a) accumulates in either thearterial wall and in vein grafts, respectively suggesting that Lp(a) canalso traverse the endothelium of arterial vessels and reach the intimaby non-receptor-mediated mechanisms and that this transport process isinfluenced by the density/size of Lp(a). There, Lp(a) can form complexeswith such tissue-matrix components as proteoglycans, glycosaminoglycans,and collagen as well as fibrin. The magnitude of the transfer of Lp(a)from the plasma compartment to the arterial wall is larger when plasmaLp(a) levels are elevated because of a gradient effect or because of apossible direct action of Lp(a) on arterial permeability.

[0466] Apolipoprotein B

[0467] Apolipoprotein B occurs in two forms termed apoB-100 and apoB-48.In humans apoB-48 is produced only by the intestine and apolipoproteinB-100 originates from the liver. Apolipoprotein B-100, which contains4536 amino acid residues, is the major apolipoprotein of VLDL, IDL,Lp(a) and is the sole apolipoprotein of LDL. ApoB-48 consists of theamino-terminal half of apoB-100, contains 2152 amino acid residues andis devoid of binding domain for the LDL receptor.

[0468] Apolipoprotein E4

[0469] Apolipoprotein (apo) E is a 34-kCa protein coded for by a gene onchromosome 19 and plays a prominent role in the transport and metabolismof plasma cholesterol and triglyceride through its ability to interactwith the low density lipoprotein (LDL) receptor and the LDL receptorrelated protein (LRP). Apolipoprotein E (apoE) is a 34-kda proteincomponent of lipoproteins that mediates their binding to the low densitylipoprotein (LDL) receptor and to the LDL receptor-related protein(LRP). Apolipoprotein E is a major apolipoprotein in the nervous system,where it is thought to redistribute lipoprotein cholesterol among theneurons and their supporting cells and to maintain cholesterolhomeostasis. Apart from this function, apoE in the peripheral nervoussystem functions in the redistribution of lipids during regeneration.

[0470] Oxidized LDL

[0471] LDL is also rapidly transported across an intact endothelium andbecomes trapped in the three-dimensional cage work of fibers and fibrilssecreted by the artery wall cells. This concentration-dependent processdoes not require receptor-mediated endocytosis. LDL entrapped inarteries or bound to receptors on endothelium or the tumormicrocirculation undergoes diverse enzymic and chemical modifications.It can also be introduced into the cell a variety of lipophilic invaderssuch as lipid peroxidation products and cholesterol oxides that mayirreversibly modify cellular functions. The early oxidative modificationof the trapped LDL in vivo occurs before monocytes are recruited andresults in the oxidization of lipids in LDL with little change in apoB.

[0472] Monocytes recruited to the lesion, are converted into macrophagesand the LDL lipids are further oxidized. Once the LDL contains fattyacid lipid peroxides, there follows (especially in the presence of metalions) a rapid propagation that amplifies dramatically the number of freeradicals and leads to extensive fragmentation of the fatty acid chainswith the generation of a broad spectrum of oxysterols, shorter-chainaldehydes (e.g., malondialdehyde and 4-hydroxynonenal) some of whichinvolve the covalent binding of short-chain substituents to the aminogroups of lysine residues in apoprotein B (and possibly to otherportions of the apoprotein B molecule) masking lysine 6-amino groups.Acetyl LDL and scavenger receptors recognize modifications effected bychemical acetylation and highly oxidized LDL.

[0473] Incubation of LDL with endothelial cells, smooth muscle cells,and macrophages in vitro induces oxidation of polyunsaturated fattyacids. Lipid peroxides formed fragment fatty acyl chains and attachcovalently to apoB or fragments thereof, thereby rendering the modifiedparticles competent for endocytosis by the scavenger receptor. LDLparticles also undergo peroxidation of polyunsaturated fatty acids whichproduces oxidative modification and conversion of LDL lecithin tolysolecithin.

[0474] Modification of LDL with malondialdehyde, a product ofarachidonic acid metabolism or oxidation of LDL leads to foam cellformation. Unlike native LDL, oxidized LDL is mitogenic or inducesapoptosis in arterial endothelial and smooth muscle cells. It alsoinduces endothelial cells and monocytes to express high levels of tissuefactor and plasminogen activator inhibitor. Levels of P-selectin areincreased intracellularly and are released by oxy-LDL which can alsodirectly stimulate PDGF production in endothelial cells. Oxidized LDLalso induce the expression of endothelin, to inhibit the expression ofnitric oxide synthase, and to inhibit the resulting vasodilation.Platelet accumulation and local increases in thromboxane A, serotonin,ADP, platelet activating factor, and activated thrombin, together with alocal reduction in prostacyclin further contribute to a procoagulantstate.

[0475] Another stable end product of cellular oxidative modification ofLDL is lysophosphatidylcholine, which is generated by phospholipase A2hydrolysis. This lipid selectively induces the expression of adhesionmolecules for monocytes, vascular cell adhesion molecule-1 (VCAM-1), andICAM-1 in cultured human arterial endothelial cells. TNF-a activation isa prerequisite for the observed lysophosphatidylcholine induction ofVCAM-1. Lysophosphatidylcholine also induces monocyte chemotaxis,arrests macrophage migration and induces macrophage proliferationthrough SR-A-mediated internalization of modified lipoprotein. Finally,lysophosphatidylcholine induces gene expression for smoothmuscle/fibroblast growth factors, the A and B chains of PDGF, andheparin-binding epidermal growth factor-like protein in culturedendothelial cells.

[0476] Oxy LDL Mimics

[0477] The cytotoxic effects of highly oxidized LDL are mimicked byhigher concentrations of oxysteroid. particularly7β-hydroperoxycholesterol. 7β-hydroxycholesterol, 7-ketocholesterol and5α-6α-epoxycholesterol. These oxysterols can induce apoptosis in avariety of cells. Of these end products,73-hydroperoxy-choles-5-en-3β-ol has been identified as the primarycytotoxic in highly oxidized LDL. This molecule accounts forapproximately 90% of the cytotoxicity of lipids extracted from highlyoxidized LDL in vitro. Fatty acid hydroperoxides and aldehydes found inoxidized LDL also alter intracellular functions. For example,4-hydroxynonenal (4-HNE). a component of oxidized LDL, induces bindingof the coagulation protein, Factor Xa to endothelial cells. In addition,oxidized LDL and mm-LDL can significantly, induce the release of IL-1from macrophages. Saponified Cu²+-oxidized LDL and mm-LDL have beenshown to contain 9-HODE, 13-HODE, and cholesterol-9-HODE; which increaseIL-1 release from macrophages. 4-HNE also causes a variety of effects onmonocytes, including stimulation of monocyte migration through inductionof chemoattractant proteins and initiation of apoptosis

[0478] Mildly Oxidized LDL (mm-LDL)

[0479] Mildly oxidized LDL (mm-LDL) induces elevated levels of cAMP by aG protein-mediated mechanism and induces inflammatory molecules both byincreasing the rates of gene transcription and by stabilizing the mRNAfor these genes. Exposure of the arterial wall to (mm-LDL) orbiologically active products of lipid peroxidation results in binding tothe LDL-R. mm-LDL also induces monocytes to bind to endothelial cells.and induces changes which affect monocyte binding, tethering,activation, and attachment. mm-LDL also induces an inflammatoryphenotype in endothelial cells and proinflammatory cytokines accompaniedby increase the levels of the transcription factor, NF kB, which hasbeen linked to the expression of a variety of adhesion molecules. Inparticular, lysophosphosphatidylcholine, a product of LDL oxidation, hasbeen shown to be a chemoattractant for monocytes and T-lymphocytes, toinduce the adhesion molecules VCAM-1 and ICAM-1, and to increase levelsof PDGF and heparin-binding epidermal growth factor mRNA in endothelialcells and smooth muscle cells. Increases in ICAM-1 expression lead toenhanced monocyte adhesion to the vessel wall.

[0480] Moreover, mm-LDL induces endothelial cells to produce the potentmonocyte activators monocyte chemoattractant protein 1 (MCP-1) andmonocyte colony stimulating factor (M-CSF). Macrophage Class A scavengerreceptors and CD36, a Class B scavenger receptor are up-regulated byM-CSF. Once bound to specific scavenger receptors, mm-LDL can initiatecell signaling events in vascular cells stimulating phosphoinositidemetabolism and calcium flux as well as stimulate phospholipase E1activity through a tyrosine kinase-dependent mechanism independent ofprotein kinase C. This induces the release of phosphatidic acid orarachidonic acid for eicosanoid production in the vessel wall. A portionof this activity may be mediated by the Class A scavenger receptorligands which stimulate macrophage urokinase expression and IL-1production a growth factor for smooth muscle cells.

[0481] The biological properties of the lipids in mildly oxidized LDLdiffer from those induced by the lipids in highly oxidized LDL. Forexample, the expression of tissue factor by endothelial cells is inducedby mildly oxidized LDL but not by highly oxidized LDL. The lipids inhighly oxidized LDL are cytotoxic, whereas the lipids in mildly oxidizedLDL are not. Mildly oxidized LDL induced the activation of the NFκB-liketranscription factor and the increase in the appearance of specificoxidized phospholipids. With continued oxidation, highly oxidized LDLsuch as lysophosphatidylcholine and oxidized sterols are produced withdifferent biological activity as given above.

[0482] The ability of mm-LDL to induce monocyte adherence to endothelialcells is mimicked by three polar bioactive lipids isolated from mm-LDLas well as oxidized 1-palmitoyl-2-arachidonyl-sn-glycerophosphocholine.The molecular structure of two bioactive lipids were identified

[0483] 1-palmitoyl-2-(5-oxovaleryl)-sn-glycero-3-phosphocholine (m/t594.3) and 1-palmitoyl-2-glutaryl-sn-glycero-3-phosphocholine (m/t610.2). The third lipid (m/t 831) has tentatively been described as anarachidonic acid-containing phospholipid containing three or four oxygenmolecules, potentially forming a conjugated triene structurecharacteristic of leukotrienes. The latter serves as a substrate forparaoxonase, and those with fragmentation products such as 5-oxyvalerateat the sn-2 position may represent substrates for PAF acetylhydroxylase.

[0484] Glycated LDL

[0485] Glycated LDL is recognized less well by the LDL receptor, but istaken up more rapidly by macrophages. Very prolonged exposure of LDL tohigh concentrations of glucose leads to glucose-mediated cross-linkingand the generation of advanced glycosylation end products, whichmacrophages recognize in a specific saturable fashion.

[0486] Artificial Complexes of LDL

[0487] Artificial complexes of LDL formed by incubation withfibronectin, heparin, and fibrillar collagen are also candidates, andthe uptake there appears to be through recognition of the fibronectin.Complexes of LDL with itself are taken up more rapidly than native LDLvia the LDL receptor. After incubation with neutrophils LDL is taken upmore rapidly by macrophages. This is attributable to the dimerization ofLDL by the action of secreted neutrophil elastase on native LDL

[0488] Apoprotein Genes

[0489] The genes for the major apoproteins associated with thelipoproteins have been cloned. These include apolipoprotein (a) (McLeanJ W Nature 330: 132-137 (1987)), apolipoprotein B-100 (Chen S H J. BiolChem. 261: 12918-12921 (1986)), apolipoprotein E4 provided by Drs. SLauer and J. Taylor. Lp(a) has been cloned from cDNA librariesconstructed from human liver mRNA (McLean J W Nature 330: 132-137(1987)). Complete sequence analysis of a 14 000-base-pair (bp) DNA copyof apo(a) mRNA showed many exact or nearly exact repeats of a 342-bpsequence occurred. Indeed, most of the mRNA consist of 22 tandem exactrepeats and 15 modified repeats. Apolipoprotein (a) belongs to a genefamily that includes genes encoding clotting factors, structuralproteins, and growth factors. Domains shared by these proteins areprotease-like domains, kringle units, calcium binding domains, andepidermal growth factor precursor domains.

[0490] In the present invention, superantigens are ligated to the majorclasses of lipoproteins in human plasma including LDL, IDL, HDL, VLDL,chylomicrons and remnants containing apoproteins and mm-LDL, oxy LDLisosterols, inositols, lysophosphatidylcholine, synthetic mimics of LDLactivity and oxyLDL byproducts by methods given in Example 47 Because oftheir unique capacity to adhere to tumor microvasculature and evoke anapoptotic/inflammatory/prothrombotic response, the lipoproteinstructures preferred for ligation to SAg include but are not limited toLp(a), LpB-1000 or B-47, oxyLDL, oxyLDL byproducts, oxyLDL mimics andIDL.

[0491] The lipoproteins used for conjugation are prepared as in Examples48-49. The superantigens used for conjugation are preferentially innucleic acid or phage form but may also be in peptide, polypeptidenucleic acid or phage display form. They are coupled to the various LDL,oxyLDL or apolipoproteins via methods given in Examples 3, 5, 47.Alternatively, SAg are incorporated or bound or conjugated to avesicular, exosomal structures shed from normal, tumor or sickled cellsexpressing LDL, oxy LDL, oxyLDL mimics or apolipoproteins.

[0492] Superantigens are also integrated into liposomal structuresprepared to express natural or synthetic LDL, oxyLDL, apolipoproteins oroxyLDL mimics as described in Section 45 and Examples 3, 5, 6, 36, 42.Optionally, integrin ligand sequences such as RGD are added tofacilitate the localization of the conjugates to the tumormicrovasculature binding to the α_(v)β₃ integrin and α_(v)β₅ integrinwhich are expressed therein (see Example 6). Thesesuperantigen-lipoprotein conjugates are physically trapped in the densenetwork of randomly branching blood vessels of the tumormicrocirculation and also bind to LDL or scavenger receptors expressedin the tumor neovasculature.

[0493] Constructs consisting of naked Sag nucleic acids containing CpGbackbone fused to apoprotein nucleic acids alone or incorporated intoliposomes are prepared as in Example 3, 6, 14, 30-31 and delivered tothe tumor sites in vivo as in Examples 14, 30-31. These constructs areuseful in vivo as a therapeutic antitumor vaccines according to Examples14, 15, 16, 18-23. They are also useful ex vivo for producing apopulation of tumor specific effector cells (T cells, γ/δT cells, NKcells, NKT) cells for adoptive immunotherapy of cancer (Examples 2-5, 7,15, 16, 18-23).

[0494] Tumor Cells or Sickled Erythrocytes and Vesicles Expressing SAgand Apolipoproteins

[0495] Superantigen nucleic acids are fused in frame to nucleic acidsencoding apoproteins including but not limited to apoproteins Lp(a),B-48 and 100 and E3 and transfected into tumor cells in vivo to producetumor cells expressing superantigens and apoproteins. These tumor cellsare recognized by apoprotein receptors in tumor microvasculature. Tumorcells are also transfected ex vivo with the identical nucleic acidconstructs. A RGD sequence is added to promote deposition in the tumormicrovasculature which are useful. These tumor cell transfectantsexpressing Sag, apoprotein and RGD bind to apoprotein receptors andintegrins respectively expressed in tumor microvasculature wherein theyinitiate a potent and localized anti-tumor response.

[0496] Superantigen nucleic acids together with nucleic acids encodingeither apo(a), apoB and apoE4 are also transfected into nucleatedsickled erythrocytes (e.g., proerythroblast or normoblast phase) bymethods given in Examples 1 and 6. The integrin ligand RGD nucleic acidsare transfected into tumor cells or sickled cells to facilitate thelocalization of the transfected tumor cells and sickled cells tointegrins expressed in the tumor neovasculature in vivo (see Example 6).Alternatively, the sickled erythrocytes or tumor cells acquire theapolipoprotein or oxyLDL by coculture with liposomes which express theapolipoprotein or oxyLDL (see Section 7 & Example 5).

[0497] These tumor cells or sickle cell transfectants are administeredparenterally and are capable of trafficking to tumor microvasculaturewherein they bind to apolipoprotein and scavenger receptors onendothelial cells and macrophages. The transfectants are phagocytosed bymacrophages cells and induce endothelial cell apoptosis. SAgs expressedon the tumor cells and sickle cells also induce a local T cellinflammatory anti-tumor response which envelops the neighboring tumorcells.

[0498] These tumor cell and sickle cell constructs are prepared bymethods given in Examples 1 & 6 and are useful in vivo against primaryand/or metastatic tumors according to Examples 14, 15, 16, 18-23.

[0499] Tumor Cells & Endothelial Transfected in Vivo with SAg andLipoprotein Receptors or Oxidized Lipoprotein Receptors

[0500] The genes encoding the LDL oxyLDL, VLDL, LRP, CD36, SREC andLOX-1 receptors as well as macrophage scavenger receptors, expressed onendothelial cells and macrophages and have been cloned. Nucleic acidsencoding receptors for various apolipoproteins including but not limitedto the LDL or apo a, apoB or apo E receptor, CD36 receptor, LRPreceptor, macrophage scavenger receptor, endothelial cell oxyLDLreceptor (LOX-1) and endothelial cell scavenger cell receptor (SREC)alone or together with nucleic acids encoding superantigens are injecteddirectly into tumor sites. The same nucleic acids are transfected intotumor cells in vivo. Transfection of these receptors into tumor cellsand tumor microvascular endothelial cells results in the expression ofthe LDL receptor protein with high affinity binding specificity for LDLoxyLDL and Lp(a). Exposure of the transfected tumor cells or endothelialcells to exogenously introduced oxidized LDL (especially sterol andlysocholinephosphatidic acid) induces tumor endothelial cell apoptosisanalogous to that seen in endothelial cell after exposure to oxyLDL. Thetransfected tumor cells internalize and degrade the oxyLDL and becausethey, like macrophages, have no means of down regulating the scavengerreceptor are transformed to “foam cells” and undergo apoptosis.

[0501] LDL Receptor (LDL-R)

[0502] The high affinity receptor for LDL known as the apoB receptor orthe LDL receptor (LDL-R) found on tumor microvascular cells as well ashepatic cells and macrophages binds LDL, VLDL and chylomicron remnantsvia their associated apoproteins. Apolipoprotein B-100 gene has beencloned (Chen S H J. Biol Chem. 261: 12918-12921 (1986)). The LDL gene ismore than 45 kilobases in length and contains 18 exons. Thirteen of the18 exons encode protein sequences that are homologous to sequences inother proteins: five of these exons encode a sequence similar to one inthe C9 component of complement; three exons encode a sequence similar toa repeat sequence in the precursor for epidermal growth factor (EGF) andin three proteins of the blood clotting system (factor IX, factor X, andprotein C); and five other exons encode nonrepeated sequences that areshared only with the EGF precursor. Tb LDL receptor appears to be amosaic protein built up of exons shared with different proteins, and ittherefore belongs to several supergene families (Sudhof T C et al.,Science 228: 815-22 (1985)).

[0503] Regulation of LDL-R expression occurs primarily at thetranscriptional level and is controlled by levels of free cholesterol inthe cell. Inflammatory mediators such as growth factors and cytokinescan promote the binding and uptake of LDL. These mediators include PDGF,TGF-b, basic fibroblast growth factor, TNFa, and IL-1. Some of thesemediators, such as TNF-a and IL-1, affect transcriptional regulation ofthe LDL-R gene at the level of the promoter.

[0504] VLDL Receptor

[0505] The VLDL receptor has been described as a new member of the LDLreceptor supergene family that specifically binds VLDL and chylomicronremnants via apolipoprotein E and lipoprotein lipase. Bothapolipoprotein E and lipoprotein lipase are constituents of chylomicronremnants, and a physiological ligand for the VLDL receptor (Niemeier Aet al., J. Lipid Res. 37: 1733-42 (1996)).

[0506] LRP Receptor

[0507] The alpha 2-macroglobulin receptor or lipoproteinreceptor-related protein (LRP) (LRP) is a cell-surface glycoprotein of4525 amino acids that functions as a multifunctional receptor whichbinds and rapidly internalizes several plasma proteins. These includealpha 2-macroglobulin-protease complexes, free plasminogen activators aswell as plasminogen activators complexed with their inhibitors, andbeta-migrating very low density lipoproteins complexed with eitherapolipoprotein E or lipoprotein lipase tissue and urokinase-typeplasminogen activators, plasminogen activator inhibitor-1, lipoproteinlipase, and lactoferrin. The active receptor protein is derived from a600-kDa precursor, encoded by a 15-kb mRNA, cloned and sequenced inhuman, mouse, and chicken. The entire human gene (LRP1) coding forA2MR/LRP has been cloned. The gene covers about 92 kb and a total of 89exons, varying in size from 65 bases (exon 86) to 925 bases (exon 89)have been identified. The introns vary from 82 bases (intron 53) toabout 8 kb (intron 6). In the introns, 3 complete and 4 partial Alusequences have been identified. Interexon PCR from exon 43 to 45 yieldeda fragment of 2.5 kb. Attempts to subclone this fragment yielded insertsranging between 0.8 and 1.6 kb. Sequencing of 3 subclones withdifferent-size inserts revealed a complex repetitive element with adifferent size in each subclone. In the mouse LRP gene this intron ismuch smaller, and no repetitive sequence was observed. In 18 unrelatedindividuals no difference in size was observed when analyzed byinterexon PCR (Van Leuven, F et al., Genomics 24: 78-89 (1994))

[0508] The LRP receptor is mainly responsible for the binding andinternalization of chylomicron remnants as well as apoE-containing HDL.ApoE-containing lipoproteins are taken up and degraded byreceptor-mediated endocytosis. Apolipoprotein E3- and apoE4-containinglipoproteins have a similar binding affinity and cause a similar degreeof lipoprotein internalization via the LDL-R and the LRP. LRP canmediate the degradation of tissue factor pathway inhibitor (TFPI), aKunitz-type plasma serine protease inhibitor that regulates tissuefactor-induced blood coagulation,

[0509] The 3 9-kDa receptor-associated protein (RAP) associates with themultifunctional low density lipoprotein (LDL) receptor-related protein(LRP) and thereby prevents the binding of all known ligands, includingalpha 2-macroglobulin and chylomicron remnants. RAP is predominantlylocalized in the endoplasmic reticulum and functions as a chaperone orescort protein in the biosynthesis or intracellular transport of LRP.RAP promotes the expression of functional LRP in vivo and stabilizes LRPwithin the secretory pathway.

[0510] Macrophage Scavenger Receptors

[0511] Scavenger receptors mediate the endocytosis of chemicallymodified lipoproteins, such as acetylated low density lipoprotein(Ac-LDL) and oxyLDL. Functional MSR are trimers of two C-terminallydifferent subunits that contain six functional domains. The MSR gene hasbeen cloned in an 80-kilobase human and localized to band p22 onchromosome 8 by fluorescent in situ hybridization and by genetic linkageusing three common restriction fragment length polymorphisms. The humanMSR gene consists of 11 exons, and two types of mRNAs are generated byalternative splicing from exon 8 to either exon 9, (type II) or to exons10 and 11 (type I). The promoter has a 23-base pair inverted repeat withhomology to the T cell element. Exon 1 encodes the S-untranslated regionfollowed by a 1 2-kilobase intron which separates the transcriptioninitiation and the translation initiation sites. Exon 2 encodes acytoplasmic domain, exon 3, a transmembrane domain, exons 4 and 5, analpha-helical coiled-coil, and exons 6-8, a collagen-like domain. Theposition, of the gap in the coiled coil structure corresponds to thejunction of exons 4 and 5. The human MSR gene consists of a Macrophagescavenger receptors (MSR) mediate the binding, internalization, andprocessing of a wide range of negatively charged macromolecules.Functional MSR are trimers of two C-terminally different subunits thatcontain six functional domains. The MSR gene has been cloned in an80-kilobase human and localized to band p22 on chromosome 8 byfluorescent in situ hybridization and by genetic linkage using threecommon restriction fragment length polymorphisms. The human MSR geneconsists of 11 exons, and two types of mRNAs are generated byalternative splicing from exon 8 to either exon 9 (type II) or to exons10 and 11 (type I). The promoter has a 23-base pair inverted repeat withhomology to the T cell element. Exon 1 encodes the S-untranslated regionfollowed by a 1 2-kilobase intron which separates the transcriptioninitiation and the translation initiation sites. Exon 2 encodes acytoplasmic domain, exon 3, a transmembrane domain, exons 4 and 5, analpha-helical coiled-coil, and exons 6-8, a collagen-like domain. Theposition of the gap in the coiled coil structure corresponds to thejunction of exons 4 and 5. The human MSR gene consists of a mosaic ofexons that encodes the functional domains. Furthermore, the specificarrangement of exons played a role in determining the structuralcharacteristics of functional domains (Emi M et al., J. Biol. Chem. 268:2120-5 (1993)).

[0512] Scavenger receptors on tumor endothelium and stroma bind oxidizedLDL, apoptotic cells, and anionic phospholipids. Class A receptors,includes the type I and II macrophage scavenger receptors (SR-M andSR-MI). They are found predominantly on macrophages and activated smoothmuscle cells. SR-M and SR-MI are homotrimeric membrane proteins, whichare derived from alternatively spliced mRNA products of a single gene.Ligands for class A receptors include acetylated LDL, oxidized LDL,fucoidan, and carrageenan. The second class, Class B scavengerreceptors, includes CD36 and SR-El, which are found in adipose tissue,lung, liver, and macrophages.

[0513] Acetyl LDL Receptor

[0514] Acetyl LDL receptor or the scavenger receptor, is distinct fromthe LDL receptor and does not recognize native LDL. It has been found ontumor microvascular cells as well as monocyte/macrophages, Kupfer'scells, and endothelial cells, particularly the sinusoidal endothelialcells in the liver. The same receptor also recognizes other chemicallymodified forms of LDL, including acetoacetyl LDL andmalondialdehyde-conjugated LDL. The acetyl LDL receptor binds OxLDL LDLmodified by incubation with cultured endothelial cells. LDL incubatedwith cultured endothelial cells for 12 to 18 hours, undergoes a physicaland chemical changes and the resulting endothelial cell-modified form ofLDL is taken up by cultured macrophages 10 times more rapidly thannative LDL. Thus, all three the major cell types in the artery wall canconvert LDL to a form recognized by the acetyl LDL receptor.

[0515] CD36 Receptor

[0516] CD36, a multigland glycoprotein structurally related to SR-BI andCLA-1 found on monocytes, endothelial cells is a high affinity receptorfor the native lipoproteins HDL, LDL, VLDL and for OxLDL and AcLDL. TheCD36 gene has been cloned (Endemann G et al, J. Biol. Chem. 268:11811-6(1993)).

[0517] Endothelial Receptors for OxyLDL: The LOX-1 Receptor (C-TypeLectin Receptor) & Scavenger Receptor Expressed by Endothelial Cells(SREC)

[0518] Endothelial dysfunction or activation elicited by oxidativelymodified low-density lipoprotein (Ox-LDL) is characterized by intimalthickening and lipid deposition in the arteries. Ox-LDL and its lipidconstituents impair endothelial production of nitric oxide, and inducethe endothelial expression of leukocyte adhesion molecules andsmooth-muscle growth factors. Vascular endothelial cells in culture andin vivo internalize and degrade Ox-LDL through a putativereceptor-mediated pathway that does not involve macrophage scavengerreceptors.

[0519] LOX-1 Receptor

[0520] LOX-1, a novel receptor for oxy-LDL, is a membrane protein thatbelongs structurally to the C-type lectin family, and is expressed invivo in vascular endothelium and vascular-rich organs. The LOX-1receptor from vascular endothelial cells has been cloned (Hoshikawa H etal., Biochem. Biophys. Res. Commun. 245: 841-6 (1998)). Mouse LOX-1 iscomposed of 363 amino acids with a C-type lectin domain type II membraneprotein structure and triple repeats of the sequence in theextracellular “Neck domain,” which is unlike human and bovine LOX-1.LOX-1 binds oxidized LDL with two classes of binding affinity in thepresence of serum. The binding component with the higher affinity showedthe lowest value of Kd among the known receptors for oxidized LDL. Withrespect to ligand specificity, LOX-1 is a receptor for oxy-LDL but notfor Ac-LDL and recognizes a protein moiety of oxy-LDL with a ligandspecificity that is distinct from other receptors for oxy-LDL, includingclass A and B scavenger receptors.

[0521] Scavenger Receptor Expressed by Endothelial Cells (SREC)

[0522] The primary structure of the SREC molecule has no significanthomology to other types of scavenger receptors, including the LOX-1receptor. The cDNA encodes a protein of 830 amino acids with acalculated molecular mass of 85, 735 Da (mature peptide). The cloned hasan N-terminal extracellular domain with five epidermal growthfactor-like cysteine pattern signatures and long C-terminal cytoplasmicdomain (391 amino acids) composed of a Ser/Pro-rich region followed by aGly-rich region (Adachi H et al, J. Biol. Chem. 272:31217-20 (1997)

[0523] The SREC mediates the binding and degradation of acetoacetylated(AcAc) and acetylated (Ac) low density lipoproteins (LDL). Isolatedsinusoidal endothelial cells from the rat liver show saturable, highaffinity binding of AcAc LDL and degrade AcAc LDL 10 times moreeffectively than aortic endothelial cells. Specific sinusoidalendothelial cells bearing the SREC not the macrophages of thereticuloendothelial system, are primarily responsible for the removal ofthese modified lipoproteins from the circulation in vivo. For thisreason, the SREC receptor and the LOX-1 receptors are preferred for usein transfecting tumor cells tumor endothelium in vivo.

[0524] Polypeptide or naked DNA encoding receptors for LDL oxyLDL, VLDL,LRP, CD36, SREC, LOX-1 and macrophage scavenger receptor (collectivelyo-LDL receptors) are used individually or together with SAg polypeptideor naked DNA containing the CpG backbone are prepared as in Examples 1,2, 3, 30-31. Alternatively, SAg are incorporated or bound or conjugatedto vesicular or exosomal structures shed from cells expressing the LDL,oxy LDL receptors. Superantigens are also incorporated into liposomalstructures which express natural or synthetic LDL, oxyLDL receptors asdescribed in Section 45 and Examples 3, 5, 6, 36, 42. All of theseconstructs are administered in vivo by any route but preferably byintratumoral injection as in Examples 2, 6, 14, 30-31. Once localized,and expressing o-LDL receptor(s) in tumor sites in vivo, lipoproteinpreparation(s) containing their respective ligands are administered tothe host. These LDL, oxyLDL or lipoproteins are non toxic to the hostgenerally but upon binding to a dense population of receptors in thetumor induce apoptosis of tumor cells and endothelial cells expressingthe receptors and initiate a well localized anti-tumor response. Thepresence of the SAg at the same site amplifies the immune andinflammatory anti-tumor effect. The advantage of this system is theminimal toxicity to the host since the o-LDL receptors are of hostorigin and the lipid infusions consist of substances which areindigenous to the host. These constructs are useful in vivo againstprimary or metastatic tumors according to Examples 14, 15, 16, 18-23.

50. SAg Inserted into Oncolytic Viral Vectors: SAg-Viral AntigenNucleotides & Fusion Proteins and Conjugates

[0525] SAgs and HPV-E6 or 7 polypeptides are chemically conjugated bymethods given in Examples 3. Alternatively, the naked nucleotidescomprising SAgs (with immunostimulatory sequences) and the HPV-E6 or E7are prepared as chimeric nucleotides or proteins by methods given inExamples 5, 30, 31 60. The SAg nucleic acids are inserted into severalsites in lytic and oncogenic viruses. For example, in HPV, the SAgnucleotide is substituted for a minimally functional domain such asviral capsid nucleotides whereas in adenoviruses, SAg is substituted forthe E1 or E3 nucleotides as in Example 60 These constructs aretransfected into tumor cells in vivo. Since HPV is not a good vector theSAg-HPV E6 or E7 chimeric plasmid is introduced preferentially intovaccinia virus for purposes of transfection. In tumor cells, the HPV E6protein triggers the inactivation of P53 via the ubiquitin pathwayfavoring the proliferation of virus and eventual tumor cell oncolysis.With viral oncolysis, the SAg nucleic acids and transcribed polypeptideare shed and the virus (and SAg nucleotides) then infect new tumor cellswhile the shed SAg polypeptide induces a T cell tumor inflammatoryresponse. Preparation of the SAg-viral constructs is given in Example60. Chimeric plasmids are prepared consisting the SAg fused to viralepitope known to be immunogenic. An example of this construct is SAg-E6or E7 construct as given in Example 60. The SAg-HPV E6 or E7 chimericgene is transfected into tumor cells as given in Examples 1 and 60. TheSAg-HPV-E6 or E7 chimeric gene, tumor cells transfected with andexpressing SAg and HPV-E6 or E7 epitopes, polypeptide conjugates orfusion proteins comprising SAgs and HPV-E6 or E7 or are used aspreventative or therapeutic vaccines under protocols given in Examples14, 15, 16, 18-23, 30, 31, 60.

[0526] DNA Vaccine Comprising a SEB Gene Linked to a to Papilloma VirusAntigen Gene (SEB-E7 and SEB-E1) Confer Protection Against TumorChallenge in Mouse and Rabbit Tumor Models.

[0527] We evaluated protective and immunotherapeutic potential ofmodified DNA vaccines by administration of fusion genes comprised ofStaphylococcal enterotoxin B (SEB) and HPV-16 E7 or CRPV E6, E7, E8(SEB-E6,E7,E8) in mouse and rabbit models.

[0528] Methods: Methods for preparation of the fusion proteins and theiradministration are given in Example 71.

[0529] We used particle bombardment with a gene gun to vaccinate C57BL/6mice and inbred EIII/JC rabbits intradermally. Mice received IIPV16-E7,vector, SEB-E7 and E7-SEB fusion genes; rabbits received CRPV E1, E6,E7, E8 genes, SEB gene, and CRPV E1, E6, E7, E8 fused to SEB. Thenanimals were challenged with HPV16 E6 and E7-containing TC-1 cells(mice) or CRPV (rabbits). In a therapeutic study, we used vector,CRPV-E7gene, SEB-E7 and E7-SEB fusion genes repeatedly at various timepoints to vaccinate outbred New Zealand white rabbits beginning 80 daysafter infection with CRPV at which time, papillomas were large andestablished.

[0530] Mouse Model: Protection with SEB-HPV16E7 Immunization

[0531] We used particle bombardment with a gene gun to vaccinate C57BL/6mice intradermally. Mice received HPV16-E7, vector, SEB-E7 and E7-SEBfusion genes. Mice were challenged with HPV16 E6 and E7-containing TC-1cells (mice) Mice receiving the SEB-E7 fusion gene showed completeprotection against challenge with TC-1 tumour cells, and remained tumourfree for 40 days. In contrast, groups of mice receiving E7-SEB, E7 only,SEB and vector all developed tumours that grew rapidly and reached 14 mmin diameter after 4 weeks. See FIG. 3.

[0532] Rabbit Model: Protection with SEB-HPV16E1 and SEB-E7 Immunization

[0533] Particle bombardment with a gene gun was used to vaccinate inbredEIII/JC rabbits intradermally. Rabbits received CRPV E1, E6, E7, E8genes, SEB gene, and CRPV E1, E6, E7, E8 fused to SEB. Rabbits werechallenged with CRPV.

[0534] The SEB-E1 fusion gene was the most effective in inhibiting thegrowth of the outgrowth of CRPV-induced papillomas. See FIG. 4.

[0535] In a second experiment, 3 of 4 vector- and 3 of 4CRPVE7-SEB-vaccinated rabbits developed malignancies 420 days after CRPVinfection. At this same time point, 5/5 rabbits receiving CRPVE7 and 5/5rabbits receiving SEB-CRPVE7 were cancer free (Table 1). The mean timeto onset of squamous carcinoma of CRPV-induced skin papillomas showedwas delayed for groups of rabbits receiving SEB-E7 and E7 only, comparedto E7-SEB and vector group (P<0.5). One of 5 rabbits receivingSEB-CRPVE7 showed therapeutic clearance of the primary papillomas (Seetable below). Rabbits with cancer/rabbits vaccinated with Day after thefollowing constructs CRPV pCX- pCX- PCX-SEB- PCX-CRPV- infection EGFPCRPV-E7 CRPV-E7 E7-SEB 338 2/4 0/5 0/5 0/4 359 2/4 0/5 0/5 1/4 389 2/40/5 0/5 2/4 423 3/4 0/5 0/5 3/4 440 3/4 0/5 1/5 3/4

[0536] Gene gun-mediated DNA vaccination by administration of fusedgenes induced strong antitumor immunity in animal models which may haveimplications in the future design of antigen-specific cancerimmunotherapy. Homologues of the papilloma antigens are alsocontemplated in this invention as fusion partners for the SAg. Likewise,the superantigen homologues may serve as fusion partners for thepapilloma antigens or homologues. Homologues that are encompassed bythis invention are given in Example.

[0537] An SE or an SE such as SEB (with Dominant Epitope Deleted)Conjugated to Papilloma Virus E7 or E1 or Other Tumor Specific Antigenssuch as the MUC1 Antigen and Costimulants OX-40 Ligand or 4-1BB1.

[0538] HPV-16 E7 is a model antigen because HPVs, particularly HPV-16,are associated with most cervical cancers. The HPV 16 E7 protein hasbeen identified as a zinc binding phosphoprotein with two Cys-X—X-Cysdomains composed of 98 amino acids. HPV-16 E7 is characterized as acytoplasmic/nuclear protein and is more abundant than E6 inHPV-associated cancer cells. Early observations analyzing HPV genomesand the viral transcription pattern in cervical carcinoma cell linesrevealed frequent integration of viral DNA and the consistent expressionof the viral early E7 gene. The same gene is necessary forimmortalization of various types of human cells. The HPV oncogenicprotein E7 is important in the induction and maintenance of cellulartransformation and is coexpressed in most HPV-containing cervicalcancers. E7 gene expression is also necessary for the proliferativephenotype of cultured cervical carcinoma cells. Therefore, vaccines orimmunotherapies targeting E7 and/or E6 proteins may provide anopportunity to prevent and treat HPV-associated cervical malignancies.While E1, E6, E7 are exemplified in this disclosure, other antigensassociated with the papilloma virus induced tumors will serve equally aswell and are contemplated as part of this invention.

[0539] SAgs and HPV-E7 or E1 polypeptide or are recombinantly fusedtogether with nucleic acids encoding the OX-40 ligand All of thecomponents of the construct are in DNA or polypeptide form. Thisconstruct is tested in murine and rabbit models of HPV infection as avaccine or against established tumors as in the above rabbit and mousepapilloma models. Immunization schedules are given in Example 71. Theconstructs are also used to treat established papillomas using the samerabbit and mouse models but administering the constructs when the tumorsare established 30-90 days after tumor implantation. The treatments aregiven parenterally by injection or infusion twice or three times weeklyfor 4-6 weeks Nucleic acid constructs are injected preferablyintratumorally twice to three times weekly for four weeks.

[0540] Dendritic Cell Ingestion of Necrotic HPV Infected Tumor Cells

[0541] Dendritic cell are exposed to SAg and HPV-E6 or E7 transfectedtumor cells which have undergone necrosis by methods including but notlimited to several cycles of freezing and thawing (Sauter B et al., J.Exp. Med. 191: 423-433 (2000)). The dendritic cells ingest the necrotictumor cell transfectants. In the dendritic cells, the viral antigensundergo cross priming to the class I pathway. These primed dendriticcells are then harvested and administered to the tumor bearing host asgiven in Examples 26-28, 60. They are also used to sensitize T cells exvivo.

[0542] In another embodiment, the DNA and RNA from these SAg-HPV-E6 orE7 transfected tumor cells or dendritic cells are extracted and utilizedfor in vivo therapy as in Examples 30-34, 60. While the HPV-E7exemplified herein is a viral immunogen, the method is applicable to anyother immunogenic viral constituent associated or etiopathogenic in themalignant process. Non-viral tumor associated antigens given inRosenberg S A Cancer: Principles and Applications of Biologic Therapy inPrinciples & Practice of Oncology sixth edition edited by V T DeVita, SHellman, S A Rosenberg, J. B Lippincott Co., Philadelphia 1999);Ostrand-Rosenberg, S. In: Gene Therapy of Cancer, E. C. Lattime et al.,eds, pp. 33-44 Academic Press San Diego, Calif. 1999; Schreiber, H., In:Fundamental Immunology, W E Paul, ed., Raven Press, New York, 1993, arealso useful. The viruses include but are not limited to adenovirus, EBvirus, herpesvirus, hepatitis B, cytomegalovirus and Kaposi's sarcoma.DNA vectors encoding SAg and other viral or non viral tumor associatedantigens are also functionally enhanced in producing an antitumor effectby the inclusion of immunostimulatory sequences, optimal promoters,introns, and polyadenylation signals. (Donnelly J J et al., Annu. Rev.immunol. 15: 617-648 (1997).

[0543] Selectivity of SAg-Viral Nucleotides for Tumor Cells

[0544] Maximum selectivity of the viral-SAg construct for tumor tissuein vivo is achieved by increasing the expression of genes necessary forreplication in tumor cells or by decreasing expression of such genes innormal cells. Approaches to express genes necessary for replication onlyin specific tissues are used. Tumor-specific viral-SAg gene expressionis accomplished through the use of tissue-specific promoters. Suchpromoters are activated preferentially in the tissue of tumor origin.Tumors arising from nonvital organs are amenable to this approach (e.g.prostate cancer or melanoma). Tissue-specific gene expression isachieved using adenovirus and retroviral vectors. The adenoviral E1Bpromoter is replaced with albumin or immunoglobulin promoters, resultingin liver- or myeloma-specific E1B mRNA transcription, respectively. TheE1A enhancer markedly increases transcription from the albumin promoter,particularly in the presence of the E1A protein. The prostate-specificpromoter and the alphafetoprotein promoter are exchanged with the E1Apromoter to achieve enhanced replication in cells from the prostate(cancer and normal cells) and in hepatocellular carcinomas,respectively. In one embodiment, the SAg nucleotide is introduced intothe above adenoviral constructs preferably at non functional sites asgiven in Example 60. The viral construct exhibits specificity for tumorcells and induces oncolysis. SAg expressed on the tumor cells induces aT cell inflammatory response. SAg polypeptide is also produced by theSAg-viral transfected bystander tumor cells that are infected with theSAg-viral construct and induces a potent inflammatory T cell tumoricidalresponse.

[0545] The preferred virus for insertion of the SAg nucleotide is anattenuated adenovirus d11520 that lacks the 827-base pair which encodesa 55-kilodalton E1B-55-kDa protein that inactivates P53. The binding ofE1B to p53 is necessary for efficient replication in normal cells.However, E1B-55-kDa is dispensable in tumor cells lacking functionalP53. Indeed, P53 function is lost in over 50 percent of all humancancers. Similarly, the deletion of specific RB-binding sites in the E1Aprotein leads to selective replication in cancer cells withabnormalities in the RB pathway and in proliferating normal cells. Suchattenuation of viruses potentially decrease replication in tumor cells.Replication competence is better maintained by selectively mutating thegene rather than deleting it completely. For example, second generationadenovirus E1B-55-kDa gene mutants still lack p53-binding but retain theviral mRNA transport function (which is lost with the total E1B-55-kDagene deletion); these viruses replicate more efficiently inp53-deficient cells than do mutants completely lacking the 55-kDa gene.SAg nucleotide is inserted into the dl1520 as given in Example 60. SAgprotein is produced by the transfected tumor cell or by bystander tumorcells that are infected by the dl1150-SAg after viral-induced oncolysis.The SAg induces a potent T cell tumoricidal inflammatory response.

[0546] An additional approach to abrogating replication in normal cells,but not in tumor cells, is to delete/mutate genes necessary forreplication in normal cells that are dispensable in tumor cells. Forexample, HSV-1 ribonucleotide reductase and thymidine kinase areessential for viral DNA synthesis in quiescent cells, whereas in rapidlyproliferating cells these enzymes are not necessary. In a clinicalsituation such as primary brain cancer the tumor cells are susceptibleto replication and lysis by such deletion mutants. In contrast, thesurrounding neurons are quiescent and resistant to viral replication.This approach results in a proliferating-cell-specific, and not acancer-specific, therapeutic agent. Nucleic acids encoding SAg areinserted into the HSV deletion mutants. SAg protein is produced by thetransfected tumor cell or by bystander tumor cells that are infected bythe SAg-HSV deletion mutants after viral-induced oncolysis. The SAginduces a potent T cell tumoricidal inflammatory response.

[0547] A class of negative selectable markers includes genes whoseenzyme protein products convert a prodrug into its toxic form. Thisclass is termed suicide genes. Genetic modification of tumor cells withthe herpes simplex virus thymidine kinase gene (HSV-TK) results inkilling of the gene-modified tumor cells by the antiviral drugganciclovir (QCV). GCV is a derivative of acyclovir, which arenucleoside analogs that can be phosphorylated by the viral thymidinekinase gene into a monophosphate form. Normal cellular enzymes can thendi- and triphosphorylate the monophosphate form into a toxic drug, whichfunctions as a DNA chain terminator and an inhibitor of the viral DNApolymerase. The HSV-TK enzyme is almost 1,000-fold more efficient atmonophosphorylating GCV than is the cellular thymidine kinase. Thus, GCVis virtually nontoxic to uninfected cells at therapeutic concentrationsof the drug (1-10 μM). Because the toxic metabolite of GCV isphosphorylated, its ability to cross cell membranes is impaired and thusits half-life within the cell is six times longer (18-24 hours) thanthat of unmodified GCV. The increased cellular half life of thephosphorylated CCV is an important feature of the anticancer effects ofHSV-TK-gene-modified tumors.

[0548] An important feature of the tumoricidal response induced bysuicide genes is that as few as 10% of the cells in a tumor containingthe HSV-TK-gene-modified cells are sufficient to kill the entire tumorpopulation. In addition long-term gene expression is not requiredbecause tumor cells expressing the HSV-TK suicide gene are killed afteronly a short exposure to GCV undergoing apoptotic or programmed celldeath initiated by the triphosphorylated toxic drug metabolite.

[0549] In this embodiment, the SAg nucleic acids are cotransfected intotumor cell together or fused to the HSV-TK gene with the HSV-TK geneafter which GCV is introduced. The SAg gene is unaffected by GCV.Expression of SAg nucleotides and polypeptides released from tumor cellsundergoing viral oncolysis enhances the primary tumor killing and thebystander effect of the suicide genes. by inducing a T cell inflammatorytumoricidal response.

[0550] Other viruses with specificity for tumor cells, proliferatingcells, ras or ras pathway activation or p53 mutations in which SAgnucleic acids are inserted include autonomous parvoviruses, reovirusesand Herpes simplex virus-1. Adenovirus (serotype 5) which is selectivefor malignant prostate cells due to E1A expression driven by a PSEelement and adenovirus (⅖ chimera) which is avid for cells lacking p53function due to a E1B-55 kD gene deletion and insertion of HSV-tk/CDfusion are also be used as vectors for superantigen nucleic acids andtargeting superantigens selectively to tumor sites.

[0551] Replication-selective adenoviruses mediate anti-tumor effects invariety of ways and are ideal vectors and devices for targetingsuperantigen nucleic acids to tumor in vivo. Adenoviruses exert directtumor cytotoxicity modulated by E3 11.6 kD and EORF4. They augmentanti-tumor immunity via CTL infiltration and direct killing (modulatedby E3gp19 kD), antigen release (modulated by E3 11,6 kD), augmentimmunostimulatory cytokine induction and increase anti-tumor cytokines(e.g., TNF, modulated by E3 10.4/14.5, 14.7 kD), enhance sensitivity tocytokines (e.g., TNF, modulated by E1A), and increase sensitization tochemotherapy. Superantigen nucleic acids are integrated into the genomeof any of the adenoviruses with the above properties and administeredparenterally, intratumorally or intrathecally to tumor bearing hosts.The superantigen nucleic acids are targeted to tumor sites where theytransfect tumor cells or non-tumor cells in the vicinity of the tumorresulting in the expression of superantigen polypeptide. In this way a Tcell inflammatory tumoricidal effect is therefore induced localizedselectively to the tumor sites.

[0552] In another embodiment, SAg nucleic acids are also inserted into aviral vector which contains a site of expression or overexpression of ahost tumor associated antigen. Nucleotides encoding tumor associatedantigens (TAAs) such as immunodominant Mart-1, tyrosinase or Mage arepreferred although any TAA nucleotide are used. These viral vector withsequences encoding SAg and tumor associated antigen retain theirspecificity for tumor cells by incorporating a tissue or tumor specificpromoter or driver encoding a key protein or transcription factor suchas prostate specific antigen, human kallikrein promoter oralphafetoprotein or host tumor suppressor binding protein. The examplesof nucleic acids encoding SAg expressed a HPV E6 or E7 or prostatespecific antigen plasmid are given above.

[0553] These SAg-viral constructs given above are transfected into tumorcells in vivo. They are given directly into tumor tissue or injectedparenterally (intramuscularly, subcutaneously, intrathecally,intraperitoneally or intravenously) where because of their selectivityfor tumor or proliferating tissue will localize in tumors. In anotherembodiment, SAg-viral naked DNA constructs are administered as a vaccineparenterally (intramuscularly, subcutaneously, intrathecally,intraperitoneally or intravenously) or directly into tumor cells invivo. Peptide or polypeptide conjugates, fusion proteins comprising SAglinked to viral epitopes and/or tumor antigens are also useful, e.g.,HPV-E6 and E7. These constructs are used as a preventative vaccines oragainst established tumors under protocols given in Examples 60.

[0554] Plasmid DNA and RNA Vectors for Production of Superantigens

[0555] To increase the potency of candidate nucleic acid vaccines, aself-replicating nucleic acid vaccine is created. The alphaviruses,accomplish self-replication through the action of a polyprotein calledRNA replicase, that is encoded within a single open reading frame.Members of the Alphavirus genus include Sindbis virus, Semliki ForestVirus (SFV) and Venezuelan equine encephalitis (VEE). Alphavirusescontain a single strand of RNA that are directly translated by ribosomes(because of its positive polarity) producing the replicase polyprotein.This polyprotein is cleaved into four subunits that can then drive notonly its own replication, but the replication of a structural proteinthat comprises the viral coat. Theoretically up to 200,000 copies of theRNAs and 100,000,000 molecules of heterologous proteins are made in asingle cell.

[0556] The activity of a replicase-containing RNA vaccine is exemplifiedin various engineered DNA plasmids which are transcribed and capped invitro. A single injection of as little as 0.1 μg the self-replicatingRNA encoding a model tumor antigen, β3-galactosidase (p-gal), issufficient to elicit antigen-specific antibody and cytotoxic Tlymphocyte responses. As little as 1 μg of the self-replicating RNAconsistently achieves complete protection from challenge with theβ-gal-expressing CT26.CL25 tumor cell line. The replicase-based RNAvaccine is therapeutically effective in mice bearing tumors injectedintravenously two days before the initiation of treatment. Tumor-bearingmice immunized with self-replicating RNA had significantly lower numbersof metastases and survived on the average 10-20 days longer than micetreated with non-replicating RNA. In addition, a several log enhancementof immunogenicity of the self-replicating vector was not merely due toenhanced production of antigen by the self-replicating RNA immunogensbut rather to the induction of apoptosis by double-stranded RNAintermediates. The self-replicating RNA enhances the presentation ofantigen by dendritic cells which occurs because of increased antigenuptake of cells that die apoptotically as a result of the activity ofthe self-replicating vector. Furthermore, double-stranded RNAintermediates formed during RNA replication induce maturation andactivation of dendritic cells, either directly, or through theelaboration of local “danger signals”. DNA plasmids using a CMV promoterwhich can also generate a long positive strand of RNA (replicon)encoding a SAg which like the alphaviral genome itself is then capableof self replication are also useful. Similarly, any RNA or DNA vectorscarrying nucleic acids encoding SAg which are enhanced by optimizingpromoters, introns, and polyadenylation signals are useful (Donnelly, JJ et al. Annu. Rev. Immunol. 15: 617-648 (1997); Leitner W W et al.,Cancer Res. 60: 51-55 (2000)). In this embodiment, SAg is substitutedfor the LacZ to produce the pRep-SEB as given in Example 60. ThepRep-SEB is used as a preventative vaccine and against established tumoras given in Example 60. There are used as preventative vaccines oragainst established tumor as given in Examples 60

[0557] Tumor Cells Transfected with SAg & SAg-Viral Constructs InduceTumor Necrosis and Enhance Tumor Antigen Presentation by DendriticCells.

[0558] SAgs are capable of inducing an necrotizing tumoricidal responseassociated with generation of cytotoxic T cells and inflammatorycytokines (Dow S W et al., J. Clin. Invest. 101: 2406-2424 (1998 ).Tumor cells transfected with SAg-viral constructs undergo oncolysis. TheSAg expressed in the construct induces a necrotizing tumoricidalresponse. Dendritic cells process the necrotic tumor cells to induce a Tcell tumoricidal effect. Macrophages phagocytosing the SAg-viraltransfected tumor cells are activated to become tumoricidal. WhenSAg-viral transfected tumor cells are exposed to exogenous apoptoticstimulus such as chemotherapy or radiation, the apoptotic tumor cellsrelease SAg nucleotides and polypeptides together with intact virus Thereleased virus spreads to infect neighboring tumor cells while the SAgpolypeptide released from the lysed tumor cells produces a T cellinflammatory tumoricidal response accompanied by activation oftumoricidal macrophages.

[0559] Tumor cells transfected with the SAg-viral constructs expressboth viral and SAg epitopes. In the case of HPV, E6 or E7 epitopes aretumor specific antigens. Exposure to SAg together with viral antigenincreases the immunogenicity of viral antigens by activating viralspecific cytolytic CD8+ cells (Coppola M A et al., Int. Immunol. 9:1393-1403 (1997). Hence, viral (tumor specific) epitopes coexpressed ontumor cells and presented with superantigen polypeptide produces anenhanced anti-viral response directed to viral epitopes expressed on thetransfected tumor cells.

[0560] Dendritic cells ingesting apoptotic or necrotic tumor cellscontaining SAg-viral nucleotides, transcribe, express and present SAgand to T cells which are activated produce a tumoricidal response. Tcells are also exposed in vitro to dendritic cells expressing SAg andviral epitopes from ingested apoptotic or necrotic tumor cells in vitro.These tumor sensitized and SAg activated T cells are used for adoptiveimmunotherapy of cancer as given in Examples 60.

[0561] The DNA and RNA from tumor cells transfected with nucleic acidsencoding SAg and viral epitopes, or dendritic cells after ingestion ofSAg transfected tumor cells are extracted and used as naked DNAconstructs for in vivo cancer therapy as in Examples 30-34, 60. Whilethe viral epitopes HPV-E-6 and E7 are exemplified herein, the method isapplicable to other viral epitopes which are known to be associated oretiopathogenic in the malignant state including but not limited toadenovirus, EB virus, herpesvirus, hepatitis B, cytomegalovirus andKaposi's sarcoma epitopes.

[0562] Adenovirus Containing Superantigen Linked to Modified Form ofIκBα.

[0563] The superantigen gene is linked the gene expressing a modifiedform of IκBα and inserted into a viral vector such as adenovirus,vaccinia or any of the well established viral vectors well known in theart and transfected into tumor cells. The modification of IκBα blocksits ability to be phosphorylated and to be subsequently degraded,thereby preventing NFκB activation and translocation to the nucleus. Inthe absence of NFκB activation exogenous TNFα induces apoptosis in theIκBα treated tumor cells. The virus also induces lysis of the tumorcells and concurrently release of the SAg polypeptides and nucleic acidsfrom tumor cells activates tumoricidal macrophages, induces anecrotizing inflammatory response which stimulates a T cell tumoricidalresponse.

[0564] SAg-viral constructs are prepared by methods given in Example 60.The SAg-viral constructs are administered intratumorally or parenterallyin the tumor models as given in Examples 60.

51. Augmented Immune Response to Cancer and Infectious Diseases:Deletion/Inactivation of Immunocyte Inhibitory Receptors and ReceptorTyrosine Based Inhibitory Motifs (ITIMs)

[0565] Many lipid-based tumor associated antigens (collectivelyLip-TAAs) consist of lipids, glycolipids, gangliosides, sphingolipidsand lipopeptides, all of which are weak immunogens and fail to evokeeffective tumoricidal immune responses. The same may be said of lipidbased antigens associated with infectious organisms. For example, tumorgangliosides on the surface or shed from tumor cells actually suppress Tcell and antigen presenting or accessory cell function. The presentinvention provides an approach directed at inhibitory receptors (IRs)and their inhibitory motifs, described below, which recognize (1)Lip-TAAs, (2) lipid-based infectious disease associated antigens(Lip-IDAs) and (3) superantigens (SAgs) or self molecules associatedwith superantigens (“SagSMs”). Deletion or inactivation of these IRs ortheir inhibitory motifs by pharmacologic or genetic means results in anenhanced cellular response to tumors or infectious organisms. The IRsspecific for Lip-TAAs, Lip-IDAs and SAgSM are abbreviated, respectively,IR_(TAA), IR_(IDA) and IR_(SAg).

[0566] Lipid antigens are presented to cells of the immune system in thecontext of their natural antigen-presenting receptors, such as CD1 orattached to lipoproteins. The present invention is designed todeactivate or eliminate the inhibitor receptors (IR_(TAA), IR_(IDA) andIR_(SAg)) on T cells, NK cells and NKT cells, all of which inhibitresponses to lipid antigens presented in the context of CD1 or asuitable surrogate, prior to exposure of these cells to a specificLip-TAA, Lip-IDA or SAgSM. The IRs recognize and respond to the sameLip-TAAs+self (e.g., CD1 or MHC) as do activation receptors with, amongother consequences, an inhibitory signal to the activation receptorafter activation receptor engagement by its ligand. The cell populationsbearing such IRs include but are not limited to T cells, NK cells andNKT cells. Antigen presenting or accessory cells (APCS) including butnot limited to macrophages, dendritic cells and fibroblasts also expressIR_(TAA), IR_(IDA)

[0567] After exposure to Lip-TAAs, a responding cell such as a T cell,NK cell or NKT cell devoid of an IR_(TAA) becomes tumoricidallyactivated due to the unopposed stimulation of activation receptors. Thisis particularly advantageous when the stimulus is a weakly immunogenicLip-TAA which binds to IRs. Similarly after exposure of APCs toLip-TAAs, a responding cell devoid of an IR_(TAA) becomes capable ofprocessing, expressing and presenting LipTAAs (and in the case ofmacrophages become tumoricidally activated) due to the unopposedstimulation of activation receptors to LipTAAs. Likewise, after exposureto Lip-IDAs, responding T cells, NK cells or NKT cells devoid ofIR_(IDA)s become cytolytic for (or otherwise destructive of) theinfectious organism bearing the Lip-IDA. Similarly, if a Lip-TAA orLip-IDA are physically associated, for example, genetically fused orchemically conjugated, to a SAgSM, after exposure to a combination ofthe SAgSM and the Lip-TAA or Lip-IDA, responding cells lacking theIR_(SAg) (and lacking IR_(TAA) or IR_(IDA)) become hyperresponsive tothese antigens.

[0568] Inhibitory Receptors (IRs) and their Motifs

[0569] Most of the IRs are members of one of two protein families: (1)Type I integral membrane proteins belonging to the Ig superfamily, and(2) Type II integral membrane proteins of the C type lectin superfamily,expressed as disulfide linked homodimers or heterodimers. These IRs areinert when self-aggregated but are able to abolish cellular signalingwhen co-ligated to activation receptors. Their cytoplasmic domainscontain one or more motifs termed “immunoreceptor tyrosine-basedinhibition motifs” or ITIMs, defined by the six-amino acid sequence(ILV)xYxx(LV) (where x is any amino acid). ITIM sequences arephosphorylated upon receptor co-ligation to create a binding site forcytoplasmic factors with SH2 domains that can transmit the inhibitorysignal intracellularly. When phosphorylated, IRs recruit one or moretypes of tyrosine phosphatases, such as SHP-1, which dephosphorylatesmolecules in the activation cascade. All the identified families of IRswith specificity for MHC class I molecules have ITIM sequences in theircytoplasmic tails.

[0570] ITIM Sequences in their Cytoplasmic Tails.

[0571] For each IR, there exists a counterpart activating receptor witha highly homologous extracellular domain and a short cytoplasmic tailthat lacks signaling capacity. The transmembrane domains of theseactivation receptors are characterized by the presence of a chargedamino acid, a hallmark of receptors that associate with accessorysubunits containing the immunoreceptor tyrosine-based activation motif(ITAM). ITIM-bearing receptors commonly inhibit activating signalstriggered by receptors with an ITAM in their cytoplasmic tails, forexample, B cell Ig receptors, TCRs, and Fc receptors (FcRs).

[0572] Cell activation mediated by ITAM-containing receptors involvesphosphorylation and activation of several tyrosine kinases andsubsequent activation of phospholipase Cγ (PLCγ) andphosphatidylinositol 3-kinase (PI3-K), together leading to theproduction of phosphoinositol messengers and a mobilization ofintracellular Ca²⁺ and a sustained increase in cytoplasmic Ca²⁺ levels.Engagement of an IR has a dominant effect, blocking the activation of anactivation receptor. In the immune system, this has effects of blockingof cytotoxicity (by T, NK and NKT cells) and cytokine production.

[0573] IRs use two different pathways to terminate cell activationdepending on the type of molecule that is recruited to thephosphorylated ITIM sequences:

[0574] (1) protein dephosphorylation mediated by tyrosine phosphatases,including SHP-1 and SHP-2, which abrogate the most proximal events inthe activation cascade resulting in the abolition of Ca²⁺ mobilization,and

[0575] (2) an inhibitory signal is generated via phosphoinositolphosphatase SHIP which hydrolyzes phosphoinositol messengers.

[0576] This latter signal does not affect proximal events triggered bythe activating receptor, such as the activation of kinases, receptorphosphorylation, or Ca²+ release from intracellular stores. Rather, itspecifically impedes extracellular Ca²⁺ influx and therefore blocks asustained increase in cytoplasmic Ca²⁺.

[0577] SHP-1, SHP-2 and SHIP, recruited by IRs, are associated withantigen-binding receptors, Fc receptors, growth factor and cytokinereceptors, and their loss, deficiency or absence results in augmentedcell activation and proliferation. These phosphatases do not functionthrough recruitment to IRs, but rather via association with stimulatoryreceptors to set thresholds or to prevent unsolicited cell activation.The dissociation of an antigen from molecules recruited by SHP-1, SHP-2and SHIP would abort a negative signal and augment cell reactivity, inparticular to antigens of tumors and infectious organisms as well as toSAgs.

[0578] Augmented Immune Response to Tumors and Infectious Agents: DualInactivation or Deletion of IR_(TAA)s or IR_(IDA)s and SAgSMs and theirITIMs

[0579] In the present invention, deactivation or deletion of IRs in Tcells, NK cells, NKT cells or APCs is accomplished either ex vivo, invivo or both, prior to exposure to the Lip-TAAs (Examples 51 and 52).This effect is achieved by several approaches, including, but notlimited to, the use of a specific antibody or antibody fragment directedto the IR(s), gene knockout, e.g., by homologous recombination exposureto an antisense nucleotide or siRNA. Deletion of IRs leads to asignificant enhancement of T cell activation in response to Lip-TAAs andSAgs. In particular, the immune cells with deleted IRs are capable ofresponding to subdominant and dominant Lip-TAAs on tumor cells,differentiating into effector cells such as CTL, and secreting cytokinesthat contribute to the tumoricidal response. The APCs deleted of IR arecapable of, processing and presenting Lip-TAAs (expressed on or shed bytumor cells) to immune cells such as T cells, NK cells or NKT cells.Macrophages deleted of IRs become tumoricidally activated.

[0580] When stimulated, the IR_(TAA), IR_(IDA), IR_(SAg) and theirrespective ITIMs inhibit cellular activation by the activation receptorsthat are specific for Lip-TAAs. The IR_(TAA)s and IR_(IDA)S on α/βTCR-bearing T cells confer specificity, respectively, for Lip-TAAs andLip-IDAs (for inhibitory signalling), optionally in the context of a CD1or a CD1 isoform. Sequence analysis of a panel of CD1-restricted,lipid-specific inhibitory TCRs on TCR positive cells reveals theincorporation of template-independent N nucleotides that encode diversesequences and frequent charged basic residues at the V(D)J junctions.The TCR CDR3 loops containing charged residues project between the CD1α-helices, contacting the lipid antigen hydrophilic head moieties aswell as adjacent CD1 residues in a manner that explains antigenspecificity and CD1 restriction.

[0581] The IR_(TAA)s respond specifically to Lip-TAAs, including fattyacids, ceramides, glycolipids, sphingolipids, glycosphingolipids,phosphosphingolipids, gangliosides, sialylated glycans and lipopeptides.IR_(TAA)s also recognize sialic acid and carbohydrates in Lip-TAAs.Sialic recognition is exemplified by Siglecs (sialic acid bindinglectins), carbohydrate recognition by some C type lectins both withITIMs in their cytoplasmic tail. The sialic acid binding region ofSiglecs is localized within the membrane-distal, amino-terminal domain.Each Siglec preferentially recognizes aspects of its sialylated ligands,such as the side chain, the N-acyl group, the linkage from the2-position, and sometimes the underlying sugars Recognition involves theentire sialic acid molecule, including the carboxylate group at the1-position, the linkage from the 2-position, the N-acyl group at the5-position, and the exocyclic hydroxyl groups at the 7-, 8-, and9-positions.

[0582] IR_(IDA)s recognize Lip-IDAs derived from bacteria, mycobacteria,parasites, fungi, protozoans or plants and respond by producing aninhibitory T cell response. Lip-IDAs comprise sphingolipids,glycopeptides, phytoglycolipids, mycoglycolipids, lipoarabinans, mycolicacids, Braun's lipopeptide, inositolphosphorylceramides and plantphosphatidylinositol. Sphingolipids with inositolphosphate-containinghead groups showing the general structure of ceramide-P-myoinositol-X(with X referring to polar substituents), includingceramide-p-inositol-mannose,inositol-1-P-(6)mannose(α1,2inositol-1P-(1)ceramide,(inositol-P)2-ceramide, inositol-P-inositol-P-ceramide andinositol-P-inositol-P-ceramide are also useful in native form ornaturally conjugated to GPI to activate T cell activation and inhibitoryTCRs.

[0583] The present invention contemplates the dual deletion orinactivation, in the same cells, of the IRs along with their respectiveITIMs. Deletion or inactivation of these IRs augments the cell'sresponses to a tumor or an infectious agents after exposure to Lip-TAAsor Lip-IDAs together with SAgSM. The lipid-based antigens are alsoimmunogenic when conjugated or fused to SAgSM or to free SAg.

[0584] Inhibitory receptors with cytoplasmic ITIMs which would beamenable to deletion of their IRTAA and ITIMs include but are notlimited to those described below.

[0585] 1. Sialic Acid Binding Lectins (Siglecs) are described in section56.

[0586] 2. Leukocyte associated Ig-like receptor-1 (LAIR-1) is a surfacemolecule expressed on human mononuclear leukocytes that functions as aninhibitory receptor on human NK cells. It is also expressed on T cells,B cells, macrophages, and dendritic cells. It is a type I transmembraneglycoprotein with a single If-like domain in the extracellular regionand a cytoplasmic tail containing two ITIMs. Cross-linking of LAIR-1 onNK and T cells delivers an inhibitory signal that decreases target celllysis. Some of the family members bind MHC class I molecules or viralMHC class I homologues. This group of receptors has not been shown tohave reactivity or specificity for Lip-TAAs.

[0587] 3. Killer cell inhibitory receptors (KIR) are a family of type Iintegral membrane protein in the Ig superfamily expressed on the surfaceof subsets of human NK and CD4+ and CD8+ T cells, predominantly CD28+memory cells. They recognize to public epitopes formed by polymorphismsat the C terminal portion of al helix of class I HLA-B and HLA-Calleles. Upon binding to HLA class I molecules KIRs prevent activationof NK cells. KIR binding to polymorphic HLA-B and HLA-C inhibitsproduction during SAg T cell stimulation (IR_(SAg)s) diminishes theproduction of SAg-induced effector T cells.

[0588] Each KIR of the Ig superfamily has two ITIM sequences (YxxL)separated by a stretch of about 26-28 amino acids. These ITIM motifs(discussed above) are critical for the inhibitory signals generated uponbinding to a class I ligand. These KIRs generate a negative signal byrecruiting and activating two protein tyrosine phosphatases containing aSrc homology region 2 (SH2) domain, SHP-1 and SHP-2. (Whereas the KIRinhibitory signal utilizes SIIP-1, the signal of another IR, FcγRII,depends on the phosphoinositol phosphatase SHIP). These tyrosinephosphatases dephosphorylate molecules in the activation cascade andthereby counter the stimulatory effects of the tyrosine kinasesassociated with activation pathways. SHP-1 binds preferentially to thesequence VxYxxL. The more N-terminal ITIM (VTYAQL) binds to both SH2domains of SHP-1, and does so more avidly than does the C-terminal ITIM(IVYELL). The N-terminal ITIM appears to be sufficient for theinhibitory signal.

[0589] 4. Dendritic cell immunoreceptor (DCIR) is a type II membraneglycoprotein with a single carbohydrate recognition domain(CRD). Itcontains ITIMs in their cytoplasmic tails. DCIRs are strongly expressedon dendritic cells, monocytes, macrophages, B lymphocytes, andgranulocytes but not on NK or T cells. These receptors do not recognizegangliosides or sialylated glycans.

[0590] Engagement of the IR has a dominant effect, blocking activation.KIRs inhibit the lytic function of CTLs activated by bacterial SAgs andregulate other T cell responses. Hence, one consequence of disrupted KIRrecognition of self-class I molecules is increased T cell cytokineproduction in response to SAg stimulation.

[0591] 5. Immunoglobulin-like transcripts (ILTs) are closely related toKIRs, bind MHC class I molecules and also contain ITIM sequences intheir cytoplasmic tails. Neither KIRs nor ILTs have been shown to havereactivity or specificity for Lip-TAAs.

[0592] 6. PD-1 is a type one transmembrane protein with an extracellularregion consisting of a single IgG-like variable (IgV) domain. It is aninhibitory member of the CD28 family. As with the other CD28 familymembers, its ligands, PD-L1 and PD-L2, are related to the B7 proteins.PD-1 is expressed on activated B and T cells, and engagement of PD-1 hasbeen shown to inhibit T cell proliferation and cytokine production inresponse to anti-CD3 and anti-CD28 antibody stimulation. Mice deficientin PD-1 expression develop autoimmune disorders characterized byproduction of high titers of autoantibodies, indicating an importantrole in the regulation of autoreactive B cells. Unlike CTLA-4, thecytoplasmic tail of PD-1 contains a immunoreceptor tyrosine-basedinhibition motif. The ITIM sequence like those found in several otherclasses of inhibitory receptors, including the killer inhibitoryreceptors found on NK cells and FcγRIIB on B cells, and functions byrecruiting SH2-containing phosphatases. There appear to be receptor andcell type specificities in the identity of the phosphatase (SHP-1,SHP-2, or SHIP) recruited by the ITIM motif. It is unknown whichphosphatase is involved in PD-1 function, although, as with CTLA-4,there is some evidence for preferential activation of SHP-2. This systemis active at low antigen concentrations inhibiting strong B7-CD28signals. Hence, this system is likely to be activated early in tumorgrowth when relatively low concentrations of TAA and IR_(TAA) areexposed to T cells resulting in the well known antigen specifichyporesponsiveness characteristic of this phase of tumor growth.

[0593] 7. B7-H1 is a cell surface glycoprotein belonging to the B7family of costimulatory molecules. It is expressed on a wide variety oftumor cells including colon, lung, ovarian and breast carcinoma afterexposure to IFN-γ. Cancer cell associated B7-H1increases apoptosis ofantigen specific T cell clones and promotes growth of tumor cells. Theapoptotic effect is mediated largely by one or more receptors other thanPD-1. It is possible that the inhibition is related to activation of anITIM sequence (as yet unidentified) that is part of the immunoregulatorycostimulation network activated by antigens presented in the context ofCD-1.

[0594] In one embodiment, this invention provides a population ofimmunocytes and APCs that are hyperresponsive to Lip-TAA and SAgs. Thesecells have been modified by functionally inactivating ITIMs (or theSH-2- or SHIP-binding sites thereof) in IR_(TAA). IR_(IDA) and IR_(SAg)using (a) pharmacologic agents, (b) specific (intracellular) antibodiesand (c) gene knockouts.

[0595] In another embodiment, the IRs are deleted or inactivated in vivoor ex vivo before exposing the cells to Lip-TAAs or SAgs by providing anantisense molecule which inactivates the relevant inhibitory ITIM orrelated signaling sequences. Cells treated in this way will then beactivated in vivo when exposed to (1) Lip-TAA expressed endogenously ontumor cells or as exogenous purified Lip-TAA preparations; (2) Lip-IDAsderived from infectious agents and expressed endogenously on theorganisms or as exogenous purified Lip-IDA preparations; or ( 3) SAg.Lip-TAAs, Lip-IDAs or SAgSM are thus used in vivo as preventive vaccinesor as therapeutic agents in the treatment of established cancer orinfectious disease (Example 54). Alternatively, IR_(TAA)S, IR_(IDA)S,IR_(SAg)S or their respective ITIMs on cells of the immune system(including T cells, NK cells, NKT cells) and antigen presenting cells(including macrophages, dendritic cells, fibroblasts) are deleted orinactivated ex vivo using methods described in Examples 51 and 52.Thereafter, these cells are exposed to Lip-TAAs, Lip-IDAs or SAgSM as inExamples 53 and 54. When it is desired to augment immune responsivenessto Lip-TAA or Lip-IDAs and SAgSM, cells are exposed to those moleculessimultaneously or sequentially as described in Example 54.

[0596] Deleterious side effects such as uncontrolled T cell activationand autoimmune responses by cells depleted of their IRs in vivo areprevented or minimized by ex vivo transduction of cells having thedeactivated or deleted IRs with the HSV thymidine kinase gene. Suchtransduced cells become susceptible to killing by ganciclovir in vivo asdescribed in Example 56, providing a “suicide” type mechanism to controlthese cells' actions.

[0597] Any IR_(TAA), IR_(IDA), IR_(SAg) can be inactivated or deleted(by the methods of Examples 51 and 52) regardless of whether it is amember of one or another recognized or new receptor superfamily orsubfamily, as long as the IR retain its functional properties andresults in augmented responses to Lip-TAAs, Lip-IDAs and SAgSM (Examples53, 54).

52. Deletion of Lip-TAA, Lip-IDA & SAgSM Motifs which Selectively Bindand Activate IR_(TAA)s, IR_(IDA)s and IR_(SAg)s

[0598] The structural motifs in the Lip-TAA, Lip-IDA and SAgSM moleculeswhich are responsible for selective binding to IR_(TAA)S, IR_(IDA)s andIR_(SAg)s and for generating an inhibitory signal, termed “antagonistmotifs,” are deleted from the Lip-TAA, Lip-IDA and SAgSM molecules. Inthis way, the remaining agonist motifs selectively bind and stimulatethe activating receptors (e.g., via activity at their ITAMs) withoutactivating the dominant IRs or their ITIMs. This results in enhancedsignaling and activation of the immune cell population in response toweakly immunogenic Lip-TAAs, Lip-IDAs and SagSM (as well as with freeSAgs.

[0599] In another embodiment, the approach of the present invention isto functionally inactivate, in a reversible manner, the molecules in theenzymatic pathway that produce activation of the IR following exposureto Lip-TAAs, Lip-IDAs or SAg, preferably the various phosphatasesdescribed above. This can be accomplished preferentially using genesilencing RNA interference molecules targeting the coding sequences ofthe IR (Example 74). Additional method s for deletion includeintracellular antibodies, gene knockout via antisense (Examples 51 and52). This leads to unopposed signaling by the activation receptor andcellular hyperresponsiveness to the lipid-based antigens, SAgSM or SAg.

53. Deletion of Nucleic Acids Encoding IR_(TAA)s, IR_(IDA)s, IR_(SAg)s,their ITIMs or Fas Genes

[0600] The present invention provides a cell of the immune system or anaccessory cell depleted not only of IR_(TAA)S, IR_(IDA)s, IR_(SAg)s butalso devoid of Fas receptors. Such a cell is (a) hyperresponsive toLip-TAAs, Lip-IDAs and SAgSM or free SAgs, and (b) resistant toapoptotic signals from tumor cells that secrete Fas ligand. Therefore,when administered to a subject, these cells manifest their tumoricidalproperties even while penetrating tumor tissue without being subject toinactivation induced by the high local concentration of gangliosidesreleased from the tumor or apoptosis due to Fas ligand secreted by thetumor cells. The deletion or functional inactivation of the Fas gene incells of the immune system, including T cells, NK cells and NKT cells,is accomplished ex vivo preferentially by RNA interference using siRNAtargeting the RNA coding sequence FAS (Example 74) or by homologousrecombination or using antisense oligonucleotides (Examples 51 and 52).These apoptosis-resistant, hyperresponsive cells are useful for adoptiveimmunotherapy of cancer (Examples 2-5, 7, 15, 16 18-23, 54).

54. Genes Encoding IR_(TAA), IR_(IDA), IR_(SAg), ITIMs and ITAMs

[0601] Genes encoding the IRs and their ITIMs are cloned usingconventional techniques. Their size and chromosomal location aredetermined using well established methods. It is predicted that theITIMs represent no more than about a 40 base pair segment. In thepresent invention, genes encoding IR_(TAA)s, IR_(IDA)s, IR_(SAg)s andtheir ITIMs including but not limited to ITIM sequences in Siglecs,LAIRs, KIRs, DCIRs, ILTs and PD-1s are deleted or inactivated inimmunocytes and APCs in vivo or ex vivo preferentially by RNAinterference targeting the coding sequences of in Siglecs, LAIRs, KIRs,DCIRs, ILTs and PD-1s (Example 74) or by antisense methodology (Examples51 and 52). These cells are then exposed to Lip-TAAs, Lip-IDAs and SAgSMor free SAgs in vivo or ex vivo (Example 53, 54). The ex vivo treatedimmune system cells are used for adoptive immunotherapy of cancer andinfectious disease (Examples 2-5, 7, 15, 16 18-23, 53, 54).

55. Therapeutic Composition: SAg or SAgSM Conjugated to Lip-TAAs andLip-IDAs

[0602] In one embodiment, SAgs are chemically conjugated to Lip-TAAs (ofthe types listed above). In another embodiment, SAgs are conjugated toLip-IDAs such as glycans and peptidoglycan antigens derived frombacteria, mycobacteria, parasites, fungi or plants. These families arelisted above. These lipid based molecules also include sphingolipidswith inositolphosphate-containing head groups as listed above. Thesemolecules are used in native form or may be conjugated to GPIstructures. They may be obtained by isolation from shed membranes,exosomes, or vesicles of the native organism. Example 55 describesextraction and purification of these lipids. SAg-Lip-TAA or SAg-Lip-IDAconjugates are prepared by methods of Examples 4-5. For immunization,these conjugates may be used alone or loaded onto soluble CD1 receptorsor onto the surface of APCs (Example 5). Example 5 also describes theseconjugates with attached GPI, fused to cell membranes, for example,tumor cell or erythrocyte membranes. In this form, the conjugates areused to activate T cells, NKT cells or NK cells.

[0603] These conjugates are used in vivo as therapeutic antitumorvaccines according to Examples 14, 15, 16, 18-23. They are also usefulex vivo for producing a population tumoricidal T cells, NK cells or NKTcells for adoptive immunotherapy of cancer (Examples 2-5, 7, 15, 16,18-23). They are used ex vivo to activate cells in which IR_(TAA),IR_(IDA) or IR_(SAg) have been deleted such as by siRNA-RNA interferencepreferentially-Example 74, gene knockout-Examples 51,52 orantisense-treatment, Example 53, 54. These hyperresponsive immune cellsare then infused into the subject using protocols set forth in Examples15, 16, 21, 23, 53, 54.

[0604] Conjugates between SAg and a Lip-IDA derived from a fungal,parasitic or mycobacterial sources are also used for the treatment ofinfectious diseases such as tuberculosis, leishmaniasis, trypanosomiasisas disclosed in Example 53. They are also useful ex vivo for activatinga population of cells in which IR_(IDA)s specific for bacterial, fungal,parasitic or mycobacterial antigens have been (1) deleted (via geneknockout) or (2) functionally inactivated (via antisense) for use inadoptive immunotherapy of infectious disease (Examples 51, 52, 53).

56. Lip-TAA Receptors and Receptor Mimics for Binding Lip-TAAs in TumorBearing Hosts Immunosuppressive Lip-TAAs

[0605] Lip-TAAs consist of sialic acid-containing glycosphingolipids;gangliosides, which are potent inhibitors of the immune response.Synthesized and shed in particularly high amounts by tumor cells, thesemolecules have been shown to enhance tumor formation and progression invivo. In the tumor cell microenvironment and ultimately in thecirculation associated with low density lipoprotein molecules, tumorgangliosides are mediators of tumor-related host immune suppression.Tumor gangliosides and glycosphingolipids (LipTAAs) are known tosuppress antigen or mitogen induced T or NK cell activation, cytotoxic Tcell generation and monocyte accessory cell function. Indeed, tumorproduction and shedding gangliosides in the course of tumor growth isone explanation for the tumor antigen specific immunosuppression whichappears within a few days after tumor implantation. For tumorganglioside induced T cell inhibition, ceramides with C16-C18 fatty acylgroups are more immunosuppressive than those with longer fatty acylchains and the presence of the sialic acid is required.

[0606] Immunocytes

[0607] LipTAAs have also been shown to modulate expression of lymphocytesurface molecules CD2, CD3, CD4, CD5, and CD8. They interact directlywith T cells to induce T cell suppression of lymphoproliferation inresponse to mitogen or antigen primed monocytes. They inhibit cytotoxiceffector function, trigger endocytosis of the CD4 molecule and interferewith the binding of IL-2 to its T cell receptors. Dephosphorylation ofthe T cell retinoblastoma protein (pRB) via phosphatase activation is anadditional mechanism of the immunosuppressive effect.

[0608] Antigen Presenting Cells

[0609] LipTAAs induce a specific, non-toxic and reversible inhibition ofthe capacity of human monocytes to induce a MIIC class II-restrictedT-cell proliferative responses in vitro. This accessory cell function isa key initial step in the induction of a cellular immune response toLipTAAs. Inhibition of accessory cell function occurs after antigenprocessing at the step of antigen localization to the APC MHC receptor.LipTAAs inhibit the production of IL-1β, TNF α and IL-6 by stimulatedadherent cells. They interfere with dendropoiesis and the expression ofcostimulatory molecules CD80, CD83, CD86 on antigen presenting cells anddendritic cells, in which case tumor associated antigens presented inthe absence of costimulation induce specific T cell tolerance.

[0610] Sialic Acid Binding Ig-Superfamily Lectins (Siglecs) Receptors,Homologues or Mimics

[0611] Siglecs (sialic acid binding Ig-superfamily lectins) are amultigene family of sialic acid binding proteins, each with a distinctspecificity for the type of sialic acid recognized and its linkage tosubterminal sugars. The family consists of sialoadhesin (siglec-1)localized to macrophages, CD22 (siglec-2) to B cells, CD33 (siglec-3) tomyelomonocytic cells, myelin-associated glycoprotein or MAG (Siglec4a)/Schwann cell myelin protein or SMP (siglec-4b) to oligodendrocytesand Schwann cells in the nervous system, Siglec-5 to neutrophils andmonocytes Siglec-6/OBBP-1 to B cells and placental trophoblasts andSiglec-7 expressed in spleen, peripheral blood leukocytes and liver.Siglec-3, 5, 6, 7 bear an extracellular sialic acid binding site and anintracellular ITIMs (tyrosine-based inhibitory motif). Siglec bindingsites for sialic acid are often masked by endogenous ligands and can beunmasked by transfection of sialyltransferases, sialidase treatment orcellular activation.

[0612] Structurally, Siglecs are a group of cell surface immunoglobulinsuperfamily lectins and a subset of the “1” type lectins which bindsialic acid. Family members each have a single V-set NH₂-terminal Igdomain followed by a variable number of C2-set Ig domains, as many as 16for sialoadhesin, or as few as one for CD33. The V- and adjacent C2-setdomains display an unusual conserved pattern of three cysteines in eachdomain. These result in an intra-□β-sheet disulfide bond in the V-setdomain, a predicted interdomain disulfide and a canonical inter-β-sheetdisulfide within the C2 set domain. The postulated interdomain disulfideis a unique feature of the siglec family.

[0613] There is significant amino acid and nucleotide sequences homologyamong the Siglec 1-7. cDNAs for all of the Siglecs are known (SeeExample 58). Siglecs 3, 5, 6 and 7 form a closely related cluster.Siglec family members CD33-or Siglec 3, Siglec 5, Siglec 6, and Siglec 7contain a conserved consensus sequence surrounding the intracellulartyrosine residues of all four of these molecules [E(I/L)xYAxL-(12-18residues)-(T/N)EYSE(I/V)(K/R)]. As with Siglec-2 and 4a, the cytoplasmictail of Siglec-3 is tyrosine-phosphorylated upon engagement. Once thetyrosines are phosphorylated, the first one is part of an ITIM(Immunoreceptor Tyrosine-based Inhibitory Motif; consensus:L/I/V/SxYxxLN), which is found in many members of the Ig superfamily(including Siglec-2/CD22), and forms a potential docking site for theSHP-1 tyrosine phosphatase. The second tyrosine-containing motif[(T/N)EYSE(I/V)] is similar to the sequence (TxYxxI/V) reported in SLAM,an immunoregulatory molecule of the Ig superfamily. This motif is thedocking site in SLAM for an SH2-containing molecule SAP (SLAM-associatedprotein), which blocks recruitment of the tyrosine phosphatase SHP-2 toits docking site in the SLAM cytoplasmic region. A similar interplayoccurs between the recruitment of phosphatases to the ITIM motif inSiglecs and the presence; of SAP or SAP-like inhibitors to the SAPbinding motif.

[0614] In contrast to the majority of immunoglobulin superfamily memberswhich recognize protein ligands, Siglec family members have all beenshown to bind to specific sialylated glycans. Each Siglec recognizesaspects of its sialylated ligands, such as the side chain, the N-acylgroup, the linkage from the 2-position, and sometimes the underlyingsugars Whereas CD22 strongly prefers glycans terminating in theSiaα2-6Gal, the other members of the family bind toSiaα2-3-Gal-terminating structures. Sn preferentially interacts withcells of the granulocytic lineage and MAG with neuronal processes.Siglec-6 has a protein ligand, i.e., leptin. For sialoadhesin (Siglec 1)and MAG (Siglec 4a), specific cell populations bearing their cognateligands are granulocytic lineage cells and neuronal processesrespectively.

[0615] The sialic acid binding property of the family is mediatedprimarily by an amino-terminal V-set Ig domain, with some contributionby the next C2-set domain. Recognition involves the entire sialic acidmolecule, including the carboxylate group at the 1-position, the linkagefrom the 2-position, the N-acyl group at the 5-position, and theexocyclic hydroxyl groups at the 7-, 8-, and 9-positions. Argininepresent in all the Siglecs is essential for sialic acid-dependentbinding. Other important interactions include hydrophobic contactsbetween conserved aromatic amino acids on the A and G B strands of the Vset domains with the N-acetyl and glycerol side groups ofN-acetyl-neuraminic acid. For Sn and CD22, the binding site has beenmapped to the GFCC′ face of the V-set domain by site directedmutagenesis. In the center of this site is an arginine residue which isfound in all members of the Siglec family. The high sequencesimilarities in this area suggests that it represents the binding sitealso in MAG, SMP and CD33. For MAG the Arg residue is necessary for thebinding of sialylated glycans.

[0616] Myelin Associated Glycoprotein (Siglec 4a)

[0617] The ligands for myelin-associated glycoprotein (MAG, Siglec 4a)are a limited set of structurally related gangliosides, known to beexpressed on myelinated neurons in vivo. The neural sialoadhesins,myelin associated glycoprotein (MAG) and Schwann cell myelin protein(SMP), had similar and stringent binding specificities. Each required anα2,3-linked sialic acid on the terminal galactose of a neutralsaccharide core, and they shared the following rank-order potency ofbinding: G_(Q1ba)>>G_(D1a)=G_(T1b)>>G_(M3)=G_(M4)>>G_(M1), G_(D1b),G_(D3), G_(Q1b) (nonbinders). Major brain gangliosides G_(T1b) andG_(D1a) adhere to MAG. The simplest ganglioside ligand for MAG is G_(M3)bearing N-acetylneuraminic acid, whereas G_(M3) bearingN-glycolylneuraminic acid did not support adhesion. The simplestganglioside ligand for MAG is G_(M3) bearing N-acetylneuraminic acid,whereas G_(M3) bearing N-glycolylneuraminic acid did not supportadhesion. G_(Q1b) alpha expressed on cholinergic neurons, is more potentthan G_(T1b) or G_(D1a) in supporting MAG-mediated cell adhesion. Analpha 2,3-N-acetylneuraminic acid residue on the terminal galactose of agangliotetraose core is necessary for MAG binding, and additional sialicacid residues linked to the other neutral core saccharides [Gal(II) andGalNAc(III)] contribute significantly to binding affinity. Binding isenhanced by pretreatment of MAG-expressing cells with neuraminidase. ForMAG, the specific cell populations bearing its cognate ligands areneuronal processes.

[0618] Sialoadhesin

[0619] Sialoadhesin had less exacting specificity, binding togangliosides that bear either terminal α2,3- or α2,8-linked sialic acidswith the following rank-order potency of binding:G_(Q1ba)>G_(D1a)=G_(D1b)=G_(T1b)=G_(M3)=G_(M4)>G_(D3)=G_(Q1b)>>G_(M1).In the case of sialoadhesin (Siglec-1), a crystal struct has beenelucidated in the presence of bound ligand, confirming that all of theseaspects of the sialic acid molecule are actually in direct contact withthe binding site. Target ligands for the sialoadhesins areglycoconjugates in which a terminal sialic acid is essential forbinding. Sialoadhesin binds predominantly to terminal α2,3-linked sialicacids, although weaker binding to α2,8-linked sialic acids wasdemonstrated. In contrast, CD22 bound only to α2,6-linked sialic acids,whereas MAG and CD33 bind only to α2,3-linked sialic acids. Amongstructures with α2,3-linked sialic acids, MAG bound preferentially to“3-0” structures (NeuAc α2,3Galβ1,3GalNAc), which are common termini ongangliosides (the major sialoglycoconjugates of the nervous system) and0-linked glycoproteins. Sialoadhesin and CD33 bound similarly to “3-0”and “3-N” (NeuAc α2,3Galβ1,4GlcNAc) structures.

[0620] CD22 (Siglec 2)

[0621] CD22 (Siglec 2) a B lymphocyte antigen preferentially binds toglycans terminating in Siaα2-6Gal and glycoconjugates terminating insialic acid. The minimal structure recognized by this lectin is thetrisaccharide Siaα2-6Galβ1-4GlcNAc, as found on N-linked, 0-linked, orglycolipid structures. One prominent ligand for CD22 is the highlyglycosylated leukocyte surface protein CD45. CD22 binds to thesialoglycoconjugate NeuAcα2-6Galβ1-4GlcNAc carried on CD45-thyN-glycans. The other members of the family bind toSiaα2-3Gal-terminating structures. Specifically, the critical featuresrequired for CD22 binding include the exocyclic hydroxylated side chainof the Sial residue and the αC2-6 linkage position to the underlying Galunit. CD22 expressed on cell surfaces is structurally and functionally amultimeric protein, demonstrating a higher apparent affinity formultiply sialylated compounds over monosialylated compounds.

[0622] CD33 (Siglec 3)

[0623] CD33 is a member of the Ig superfamily that is restricted tocells of the myelomonocytic lineage but whose functions and bindingproperties are unknown. It shares sequence similarity with sialoadhesin,CD22, and the myelin-associated glycoprotein which constitute thesialoadhesin family of sialic acid-dependent cell adhesion molecules.CD33 is a fourth member of this family. A recombinant soluble form ofCD33, Fc-CD33, bound red blood cells with a specificity similar to thatof sialoadhesin, preferring NeuAc2,3Gal in N-and 0-glycans overNeuAcα2,6Gal in N-glycans. Pretreatment of the CD33-transfected cellswith sialidase rendered them capable of mediating sialic acid-dependentbinding. CD33 can function as a sialic acid-dependent cell adhesionmolecule and that binding can be modulated by endogenoussialoglycoconjugates when CD33 is expressed in a plasma membrane.

[0624] The Siglec polypeptide family of receptors, homologues or mimicsare prepared from their cDNAs using recombinant techniques as in Example58. These receptors are used to bind circulating immunosuppressiveLip-TAAs in vivo. They are expressed as part of a chimeric receptor withadditional molecules such as Fc fragments which facilitate the removalof Lip-TAAs from the tumor bearing host. They are also useful whenadministered in advance of T cells, NK cells or APCs in which IR_(TAA),IR_(IDA) inhibitory receptors have been deleted ex vivo (e.g., via geneknockout) or inactivated (e.g., via antisense) Example 58.

[0625] Additional Lip-TAA Binding Molecules:

[0626] Prosaposin (SAP precursor) exists as an integral membrane proteinbound by a mannose-6-phosphate-independent mechanism. Prosaposin is theprecursor or four smaller lysosomal saposin A, B C and D proteins. Bothprosaposin and mature saposins interact specifically with glycolipidsand serve as nonenzymatic cofactors in the hydrolysis ofglycosphingolipids by acidic glycosidases. As a family they are known assphingolipid activator proteins. Prosaposin is the only member of thesaposin family with an extralysosomal location existing as a membranebound or secretory protein. It exhibits ubiquitous expression,evolutionary conservation, and diverse tissue inducibility.

[0627] The function of the secreted form of prosaposin is as aglycolipid binding and transfer protein in vivo. Prosaposin and saposinsare capable of transferring gangliosides from donor liposomes toacceptor membranes including erythrocyte ghost membranes. With its fourlipid binding domains prosaposin forms clusters with certainsphingolipids in the plasma membrane and regulates their endocytosis anddegradation. Prosaposin, but not mature SAPs, has been detected inhumansecretory fluids like cerebrospinal fluid, semen, milk, pancreaticjuice, and bile. In rat tissues, skeletal muscle, heart, and braincontain mainly precursor forms, while mature forms dominate in spleen,lung, liver and kidney.

[0628] Prosaposin and saposins A, B, C, and D form stable complexes with13 different gangliosides. Gangliosides of the gangliotetraose type(“a”) series) bind with high affinity, whereas (“b”) seriesgangliosides, 0-acetylated gangliosides, and gangliosides with shortercarbohydrate chains, bind with lower affinity. Several gangliosides (“a”series) bind to prosaposin with high affinity forming stableganglioside-prosaposin complexes.

[0629] The derived amino acid sequence of SAP precursor is given inO'Brien J S et al, Science 241: 1098-1101 (1988). Amino acids 229 to 323are colinear with the chemically sequenced amino acids of SAP-2. Thenucleotide sequence from which the protein sequence was derived isdeposited in the GenBank (access code J03086). The four SAP-like domainsare aligned to emphasize the nearly identical placement of cysteineresidues, glycosylation sites, and conserved proline residues.

[0630] Prosaposin cDNA has been elucidated by O'Brien J S et al.,Science 241: 1098-1101 (1988). The open reading frame contains regionscoding for mature SAP-1 and SAP-2 and the four SAP-like domains. Domains1 to 4 correspond to amino acid residues 47 to 130, 182 to 262, 298 to377, and 392 to 474, respectively. The prosaposin gene covers a regionof at least 17 kb on segment q21 of chromosome 10. The positions of its14 introns, the cysteine residues and the N-glycosylation sites showsome regularities supporting the assumption that the sap-precursor genearose by sequential duplication of an ancestral gene coding for only onesap. Exon 8 consists of only nine base pairs which code for three aminoacids near the C-terminus of sap-B. In different human cDNA libraries,cDNAs are found with or without the nine bases of exon 8. Afteramplification by polymerase chain reaction and direct sequencing, thesetwo forms and a third one containing only the last six bases of exon 8are detected in cDNAs prepared from cultured human skin fibroblasts. Allthree forms are found in cells from a normal proband and from twopatients with sap-B deficiency due to a point mutation in exon 7. ThecDNA of prosaposin without the nine extra bases of exon 8 encodes apolypeptide chain of 524 amino acids starting with a typical signalpeptide or amino acids for entering the ER.

[0631] The prosaposin cDNA construct is also generated by amplifying arat testicular Zap cDNA library by PCR (polymerase chain reaction) withtwo synthetic oligonucleotide primers. A positive signal of 1.7 KB wasisolated, subcloned into the pGEM-7Zf(+). Sequence analysis showed anear-identical nucleotide and amino acid similarity to a previous ratprosaposin cDNA.

[0632] The nucleic acids encoding Siglecs, prosaposin, saposins or otherLip-TAA binding proteins are inserted into the RNA (or DNA) replicatingvector to create a pRcp-Lip-TAA binding protein. This construct isadministered in vivo, e.g., intratumorally, subcutaneously orparenterally wherein it produces the Lip-TAA binding protein which bindsto Lip-TAAs produced or shed by tumor cells and prevents them fromexerting their immunosuppressive effects on host T cell and antigenpresenting cell function.

[0633] Saposins

[0634] Saposins are a family of four small glycoproteins, all of whichare derived from prosaposin, and are involved in the lysosomalhydrolysis of various sphingolipids. They are water soluble and highlyhydrophobic. Saposin B binds to complex gangliosides (such as G_(M1) andG_(D1a)) and transports them from donor to acceptor liposomes.Prosaposin and all the saposins bind gangliosides, sulfatides, andcerebrosides to varying degrees and facilitate glycolipid insertion intoerythrocyte ghosts or brain microsomes to differing extents. Also, thesulfatide activator protein (SAP A) markedly accelerates the transfer ofcomplex gangliosides compared with simpler glycolipids having shortercarbohydrate chains.

[0635] The amounts of ganglioside bound in vivo depends on many factors,including the state of the ganglioside (micellar or membrane bound), thetype of ganglioside, the pH of binding, the presence of divalentcations, other lipids, and other binding proteins. The highest affinityof prosaposin (and saposins A, B, C, and D) is for the “a” seriesgangliotetraose-type gangliosides possessing terminal sialic acidresidues. Ganglioside G_(Q1b) has a unique potency to enhanceneuroblastoma cell proliferation and to increase the number and totallength of neurites (neuritogenesis) in vitro. This ganglioside is boundtightly by prosaposin.

[0636] Although one SAP, i.e., G_(M2) activator protein, has been shownto display in vitro glycolipid transfer activity, this SAP is clearlyselective for ganglioside G_(M2) Other SAPs (four of five) are encodedby a single gene and enter the lysosome as a single 65-73-kDa precursorchain that is processed into four similarly sized (˜13 kDa), fairlyhomologous polypeptides (SAPs A-D) that are heavily glycosylated andhave acidic pI values.

[0637] Homologies among the saposins range between 16% (saposinB/saposin C) and 39% (saposin A/saposin C) with 15 amino acids beingconserved in all four proteins, among them all six cysteine residues andan N-glycosylation signal. When placement of amino acids with similarproperties at the same position in each sequence is considered, thesimilarity among saposins increases to between 47% (saposin B/saposin C)and 60% (saposin A/saposin C).

[0638] Models of each saposin indicate a compact rigidly cross-linkeddisulfide-bridged polypeptide containing a common hydrophobic pocket,suggesting that they should all act as lipid binding proteins.

[0639] cDNAs encoding for SAP-1 and SAP-2 have been isolated (O'Brian JS et al., Science 241: 1098-1101 (1988)). The nucleotide sequence of thelargest cDNA is colinear with the derived amino acid sequence of SAP-2and with the nucleotide sequence of the cDNA coding for the70-kilodalton prosaposin (prosaposin cDNA). The coding sequence formature SAP-2 is located 3′ to that coding for SAP-1 in the prosaposincDNA. Both SAP-1 and SAP-2 are derived by proteolytic processing from acommon precursor that is coded by a genetic locus on human chromosome10. Two other domains similar to SAP-1 and SAP-2 are also identified inprosaposin. Each of the four domains is approximately 80 amino acidresidues long, has nearly identical placement of cysteine residues,potential glycosylation sites, and proline residues. Each domain alsocontains internal amino acid sequences capable of forming amphipathichelices separated by helix breakers to give a cylindrical hydrophobicdomain that is stabilized by disulfide bridges. Protein immunoblottingexperiments indicate that prosaposin (70 kilodaltons) as well asimmunoreactive SAP-like proteins of intermediate sizes (65, 50, and 311kilodaltons) are present in most human tissues.

[0640] Glycolipid Transfer Protein

[0641] Glycolipid transfer protein (GLTP), a nonglycosylated,cytoplasmic protein is present in most animal cells, and is acytoplasmic protein. The purified protein has a molecular weight near 23kDA and has an isoelectric point near 9.0. It contains 209 amino acidsresidues a high level of amino acids with non-polar side chains (48%).Northern blot analysis of shows a single transcript of 2.2 kilobases andthe following hierarchy of RNA levels: cerebrum>kidney>spleen congruentwith lung congruent with cerebellum>liver>heart muscle. The amino acidsequence of a glycolipid transfer protein from pig brain has beendetermined by automatic sequencing and fast atom bombardment massspectroscopic analysis of peptides produced by chemical and enzymaticcleavage reactions. The protein consists of 208 residues, withN-acetylalanine as the N-terminal residue and valine as the C-terminalresidue. It contains 3 cysteine residues. The primary structure of theglycolipid transfer protein from pig brain is as follows: Acetyl-A-L-L-A-E-H-L-L-K-P-L-P-A-D-K-Q-I-E-T-G-P-F-L-E-A-V-S-H-L-P  30P-F-F-D-C-L-G-S-P-V-F-T-P-I-K-A-D-I-S-G-N-I-T-K-I-K-A-V-Y-D  60T-N-P-A-K-F-R-T-L-Q-N-I-L-E-V-E-K-E-M-Y-G-A-E-W-P-K-V-G-A-T  90L-A-L-M-W-L-K-R-G-L-R-F-I-Q-V-F-L-Q-S-I-C-D-G-E-R-D-E-N-H-P 120N-L-I-R-V-N-A-T=K-A-Y-E-M-A-L-K-K-Y-H-G-W-I-V=Q-K-I-F-Q-A-A 150L-Y-A-A-P-Y-K-S-D-F-L-K-A-L-S-K-G-Q-N-V-T-E-E-E-C-L-E-K-V-R 180L-F-L-V-N-Y-T-A-T-I-D-V-I-Y-E-M-Y-T-K-M-N-A-E-L-N-Y-K-V-OH 208

[0642] The purified GLTP is a mixture of two forms of a protein; one ofthem (about 15%) with an intramolecular disulfide bond, and a reducedform (about 800) without one. GLTP with an intramolecular disulfide bondhas twice as much activity as the reduced form, and formed moreGLTP-glycolipid complex. GLTP is distinctly different from other solubleproteins that interact with glycolipids such as other types lipidtransfer proteins, and lectins with mannose recognition/binding domains.

[0643] GLTP is specific for neutral glycosphingolipids and gangliosidesbut not phospholipids or neutral lipid intermembrane transfers. Theprotein transfers, by a carrier mechanism, glycolipids with a β-glucosylor β-galactosyl residue directly linked to either ceramide ordiacylglycerol hydrophobic backbone. It has been shown that GLTPfacilitates the transfer of glycolipids only when both the donor andacceptor membranes are in the liquid-crystalline state. TheGLTP-glycolipid complex is formed as a result of removal of a glycolipidmolecule from a membrane and binding to a GLTP molecule. GLTP from pigbrain facilitates the transfer of glycosphingolipids which includeglucosylceramide, galactosylceramide, lactosylceramide, sulfatide.lactosylceramide-II-sulfate, globotriaosylceramide,globotetraosylceramide, globopentaosylceramide,sialosyllactosylceramide, and GM₁ ganglioside. The transfer ofManβ1-4Glcβ1-ceramide and Manα1-4Manβ1-4Glcβ1-ceramide is alsofacilitated by GLTP from pig brain. In addition to glycosphingolipids,GLTP from pig brain also facilitates the transfer of3-[Galβ1]-sn-1,2-diacylglycerol, 3-[Galα1-6Galβ1]-sn-1,2-diacylglycerol, and3-[Glcβ1]-rac-1-2-dipalmitylglycerol. The protein does not facilitatethe transfer of 3-[Manα1-3Manα1]-sn-1,2-diacylglycerol,3-[Glcα1]-sn-1,2-diacylglycerol, phosphatidylcholine,phosphatidylinositol, phosphatidylethanolamine, cholesterol, orcholesteryl oleate. GLTP from pig brain stimulates the transfer ofpyrene-labeled sphingomyelin to a very small but significant extent.GLTP recognizes and binds the sugar residue directly linked to eitherceramide or diacyglycerol in addition to the nonpolar portion ofglycolipids.

[0644] The cloned bovine and porcine brain cDNAs for GLTP contain 627-bpopen reading frames. The encoded amino acid sequences in the full-lengthbovine and porcine cDNAs are identical, consisting of 209 amino acidresidues, Expression of GLTP-cDNA in Escherichia coli using pGEX-6P-1vector results in glutathione S-transferase-GST-GLTP fusion protein with50% of expressed fusion protein being soluble and active. Proteolyticcleavage of GSTGLTP fusion protein (bound to GST-Sepharose) and affinitypurification resulted in fully active GLTP. Northern blot analyses ofbovine tissues showed a single transcript of ˜2.2 kilobases and thefollowing hierarchy of mRNA levels: cerebrum>kidney>spleen, lung,cerebellum>liver>heart muscle.

[0645] Non-Specific Lipid Transfer Proteins (nsLTPs)

[0646] Nonspecific lipid transfer proteins (nsLTPs) are present in bothanimals and plants and represent a group soluble proteins that catalyzethe in vitro transfer of a wide range of lipids, including glycolipids.Although able to transfer different neutral glycosphingolipids andganglioside G_(M1) between membranes, nsLTPs clearly differ from GLTPwith regard to lipid selectivity and ability to actively transfer allcommon phosphoglycerides as well as cholesterol. Also, despite havingbasic isoelectric points (pI 8.6-9.6) like GLTP (pI 9.0), nsLTPs aremuch smaller (13.2 kDa) than GLTP (23.9 kDa) and are initially encodedin precursor form (15.3 kDa; pre-nsLTP). A tripeptide targeting sequence(Ala-Lys-Leu) in the C-terminal end directs certain nsLTPs toperoxisomes, where a 20-amino acid presequence is cleaved. No knownorganelle targeting sequences appear in the primary sequence of GLTP,which is consistent with its putative cytoplasmic localization. Giventhe differences in selectivity and structure along with the lack ofsequence homology revealed by searches on-line data bases, availableevidence indicates that GLTP is distinctly different from known nsLTPsand other lipid transfer proteins.

[0647] A non-specific transfer protein isolated from beef liveraccelerates the exchange and net transfer of phospholipids andcholesterol between natural and artificial membranes (Crain R C et al.,Biochemistry 19:1433-1439 (1980)). The bovine liver protein transfersphosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol,sphingomyelin, phosphatidic acid, phosphatidylserine,phosphatidylglycerol, and cholesterol and also promotes net transfer ofphospholipids from multilamellar vesicles to human high densitylipoproteins or from small unilamellar vesicles to mitoplasts. Thisprotein also accelerates the net transfer of cholesterol from smallunilamellar vesicles to multilamellar vesicles.

[0648] Bloj et al., J. Biol. Chem. 256:5988-5991 (1981) demonstratedthat this protein also accelerates the transfer of labeledglycosphingolipids between membranes. The protein shows littlespecificity toward the polar head of complex lipids and is known totransfer the sphingophospholipid, sphingomyelin.Di-palmitoylphosphatidylcholine is transferred 2.6 times faster thanglobotetraglycosylceramide from egg phosphatidylcholine vesicles toerythrocyte ghosts. Crain et al., Biochemistry 19:1433-1439 (1980))noted that phosphatidylcholine transfer was 3 times faster than that ofsphingomyelin. Glycerophospholipids are better substrates thansphingolipids for the lipid transfer protein. The nonspecific lipidtransfer protein is the first protein shown to move complex neutralglycosphingolipids and gangliosides between membranes. In vivo suchproteins serve to facilitate the transfer of glycolipids from theirsites of initial synthesis to other sites where glycosylation may takeplace or where the glycolipids express their biological functions.

[0649] Additional Ganglioside Binding Proteins

[0650] Ganglioside binding proteins especially in brain or culturedneuronal cell lines are membrane-associated and specifically bindgangliosides of the “b” series with high affinity. This protein islocalized to myelin membrane preparations; it differs from prosaposin inboth specificity and localization. In cultured cerebral granular cellsseveral membrane proteins bind photoreactive ganglioside G_(M1)derivative. Two prominent proteins so-labeled are released by trypsinand their molecular masses (46 and 31 kDa) are similar to the initialproducts of proteolysis of prosaposin. However, additional bindingproteins varying from 12 to 112 kDa are also present and otherganglioside binding proteins that have basic isoelectric points.

[0651] Two glycolipid transfer proteins isolated from calf braincatalyze the transfer of gangliosides and neutral glycosphingolipidsfrom phosphatidylcholine vesicles to erythrocyte ghosts (Gammon C M etal., Biochemistry 26: 6239-6243 (1987)). Purification by differentialcentrifugation, ammonium sulfate precipitation, and gel filtration andfast protein liquid chromatography (Mono S), produced two peaks ofactivity. The major band of both fractions with a molecular mass ofapproximately 20,000 daltons catalyzed the transfer of ganglioside GM 1as well as asialo-G_(M1), but transfer protein I (TPI) is more effectivewith di- and trisialogangliosides. Transfer protein II (TPII)is morespecific for neutral glycolipids in that G_(A1) is transferred morerapidly than any of the gangliosides; however, lactosylceramide transferis relatively slow. Neither protein catalyzed transfer ofphosphatidylcholine.

[0652] The specific transfer activity toward additional glycolipidsubstrates. revealed TPI to be more reactive toward G_(M1) than G_(A1),its asialo derivative. The other gangliosides are only slightly lessreactive while Lac-Cer is the least effective substrate. A differentorder of reactivity is seen for TP II: G_(A1) was transferred somewhatmore efficiently than GM1, and the other gangliosides are transferred atconsiderably lower rates. Neither protein catalyzes the transfer ofphosphatidylcholine. TPI, with an apparent preference for gangliosidesover neutral glycolipids of shorter chain is a different protein fromGLTP. The fact that neither this transfer proteins nor GLTP catalyzedtransfer of phospholipids indicates a basic difference from the transferprotein of bovine liver described by Bloj et al., J. Biol. Chem.256:5988-5991 (1981).

[0653] The activator protein which promotes hexosaminidase A catalyzedhydrolysis of G_(M2) and G_(A2) also functions as a ganglioside transferprotein in the absence of enzyme (Conzelmann W et al., Eur. J. Biochem.123:455-464 (1982)). The activator protein extracts glycolipid monomersfrom micelles or liposomes to give water-soluble-complexes with astoichiometry of 1 mol of glycolipid/mol of activator protein. In theabsence of enzyme the activator acts in vitro as glycolipid transferprotein, transporting glycolipids from donor to acceptor membranes. Theactivator protein is specific for ganglioside G_(M2). Other glycolipids(G_(M3). G_(M1). G_(D1a) and G_(A2)) form less stable complexes with theactivator and are transferred at a slower rate (except for gangliosideG_(M1)) than ganglioside G_(M2).

[0654] In the present invention, Lip-TAAs receptors on T cells, α/β+ orαβ--T cells, γ/δT cells, NKT cells, NK cells and/or antigen presentingcells of any type and dendritic cell used individually or collectivelyin any combination are inhibited deleted or mutated (to functionallydeactivate them) in vivo using antisense techniques given in Examples51, 52 or preferentially RNA interference as given in Examples 18-20,74. These receptors are tested in vivo in animal models as given inExamples 18-20, 51, 52, 57-59, 74. Ex vivo deletion, inhibition,mutation of LipTAAs receptors on immunocytes or accessory cells iseffected by antisense or gene knockout, ribozyme knockout techniques(Examples, 51, 57-60) or preferentially RNA interference (Examples18-20, 74). These cells with deleted or inactivated LipTAAs receptorsare incubated with Lip-TAAs ex vivo for 24 hours. They are alsoeffective without specific ex vivo antigen stimulation assuming thatthey will encounter the specific Lip-TAA in vivo. They are useful inconventional models of tumor outgrowth and established tumors as welllas adoptive immunotherapy protocols given in Examples 18-20, 57-59, 68.Prior to the adoptive transfer of IR_(TAA) depleted and activatedimmunocytes or APCs (1-4 hours is preferred but 24-72 hours isacceptable), tumor bearing hosts are treated with decoy LipTAA receptorpolypeptides to prevent circulating LipTAAs associated with serumlipoproteins from tolerizing the infused immunocytes or accessory cells(Examples 57-59).

[0655] In another embodiment, purified LipTAAs receptor or transferprotein genes are used to prepare purified LipTAAs receptor or transferpolypeptides. These structures are useful as intact polypeptides,fragments or chimeric (e.g., fused to Fc fragment) proteins withconserved Lip-TAA binding domains. These purified receptors, fragmentsor chimeras are useful as decoys in vivo to bind Lip-TAAs in vivo thuspreventing them from exerting their immunosuppressive effects onimmunocytes and accessory cells. They are prepared in soluble form orthey are incorporated into phage displays or liposomes and used in vivoin tumor systems given in Examples 57-59.

[0656] Similarly, the present invention contemplates thereduction/deletion of immunosuppressive ganglioside production/secretionin ganglioside-producing tumor cells such as neuroblastoma-tumor cells.Production of mono-, di- and tri-sialo gangliosides G_(M1) G_(M2),G_(M3), G_(D1a,) G_(D1b), G_(D3), GT_(1a), G_(T1b) production isinhibited by introduction of silencing, inhibitory siRNA specific forsequences encoding ganglioside glucosyltransferases, gangliosidegalactosyltransferases or ganglioside sialyltrasferases in tumor cells(Example 74). Other ganglioside transferases active in the synthesis ofauthentic or incomplete gangliosides with substantially the samefunction as the native gangliosides may also be targeted by siRNAs.Human or mouse neuroblastoma cell lines, e.g., human SK—N-BE and murineNeuro-2a NB cells are used to induce tumors in SCID mice. Transfectionof siRNA specifie for the coding regions of gangliosideglucosyltranferases, ganglioside galactosyl transferases or gangliosidesialyltrasferases in tumor cells is accomplished ex vivo prior to tumorcell. implantation as given in Examples 1 and 74. The outgrowth oftumors is monitored in the siRNA transfected and mock transfected tumorgroups. Tumor outgrowth is minimal in the mice injected with tumor cellstransfected with the siRNA whereas mock transfected tumor cells growrapidly and kill the mice within 28 days of implantation. In this way,the powerful immunosupressive effects of tumor gangliosides on T cellsand antigen presenting cells is minimized or eliminated.

57. Deletion of Nucleic Acids Encoding Src-Like Adapter Protein (SLAP)SLAP Protein

[0657] The Src-like adaptor protein (SLAP) contains Src homology (SH)3and SH2 domains, which are highly homologous to those of Lck and otherSrc family members. Like Lck. this Src-like adaptor protein (SLAP)contains a short unique NH,-terminal domain, followed by SH3 and SH2domains. However, it does not contain a tyrosine kinase domain. Insteadof a kinase domain. SLAP contains a unique COOH terminus of 104residues. Both the SH3 and SH2 domains of SLAP are highly homologous tothose of Lck and share with them 55 and 50% identity, compared with 42and 43% with c-Src, respectively A structure-function analysis revealedthat both the SH3 and SH2 domains contribute to this inhibitoryfunction. SLAP was cloned using a yeast two-hybrid screen (21).

[0658] SLAP is expressed in T cells. When expressed in Jurkat T cells itcan specifically inhibit TCR signaling leading to nuclear factor ofactivated T cells (NEAT)-, activator protein I (AP-1)-, and interleukin2-dependent transcription. The SH3 and SH2 domains of SLAP are requiredfor maximal attenuation of TCR signaling. SLAP acts proximally in theTCR signaling pathway. In stimulated Jurkat cells, SLAP associates witha molecular signaling complex containing CD3ζ, ZAP-70, SH2domain-containing leukocyte protein of 76 kD (SLP-76), Vav, and possiblylinker for activation of T cells (LAT). Collectively, these resultssuggest that SLAP interacts with the stimulated TCR complex todownregulate TCR-mediated signals by interfering with the proximalsignaling events. Therefore, the differential expression of SLAP proteinin T cells regulates sensitivity to TCR stimulation, thus influencingmature T cell responses to antigenic stimulation.

[0659] The present invention contemplates the deletion of inhibitoryadaptor proteins such as SLAP from T cells in tumor bearing hosts usingantisense techniques in vivo as given in Example 51. Alternatively, SLAPdeletion can be achieved ex vivo in a population of T cells includingγ/δ T cells, α/β T cells using gene knockout or antisense techniques asgiven in Examples 51-52. These T cells are harvested and used for theadoptive immunotherapy of cancer as given in Examples 15, 16, 21, 23,68.

58. Superantigen Conjugated to Thrombospondin I and Type 1 RepeatPeptides of Thrombospondin and Other Molecules Inducing Apoptosis ofEndothelial Cells

[0660] In the present invention, superantigens are conjugated tomolecules which induce apoptosis of endothelial cells. Superantigens areknown to induce tissue inflammation and in the absence of effector cellsor their mediators are capable of inducing endothelial cell injury. Theyare conjugated to thrombospondin 1 and type 1 repeat peptides ofthrombospondin which also induce apoptosis of tumor neovascularendothelial cells. The native sequence KRFKQDGGWSHWSPWSSC or themodified sequence which lacks the TGF, activating sequenceKRAKAAGGWSHWSPWSSC equally stimulated DNA fragmentation. The basicresidues and the WSXW motif are both required for optimal activity. Thethrombospondin and/or the superantigen in the conjugate may be used innucleic acid form. The superantigen polypeptide or nucleic acid in theconjugate is capable of evoking an inflammatory response in the tumormicrovasculature while the thrombospondin induces apoptosis of tumorendothelial cells. Conjugations of polypeptides and/or nucleic acids arecarried out by methods given in Example 4 and 5. The conjugates areprepared by chemical crosslinking using homo or heterobifunctionalcrosslinking agents, carbodiimide, cyanoborohydride or glutaraldehyde asgiven in Hermanson G. Bioconjugate Technology Academic Press, New York,N.Y. (1996). They are also prepared as fusion proteins or phage displaysaccording to protocols given in Examples 5 and 6. They are also usefulin nucleic acid form as a chimeric SAg-thrombospondin nucleic acidconstruct since the CD36 gene has been cloned (Wyler B et al., Thromb.Hemost. 70: 5001505 (1993); Armsella A L et al., J. Biol. Chem. 269:18985-18991 (1994). The conjugates are useful as a preventative ortherapeutic anti-tumor vaccine according to Examples 15, 16, 18-23. Theyare also used ex vivo to produce tumor specific effector cells foradoptive immunotherapy of cancer (Examples 2-5, 7, 15, 16, 18-23).

59. Removal of SE-Specific Antibodies with Anti-Idiotype Antibodies

[0661] It is well established that naturally occurring antibodiesspecific for SEs are present in a large percentage of human patients.During clinical trials using a fusion protein of SEA conjugated to atumor specific antibody, antibodies specific for SEA appeared in theserum. The titer of these antibodies often rose with treatment and theirappearance correlated with increased toxicity. These antibodies alsointerfere with the ability of the SE conjugates to induce tumoricidaleffects by binding to the T cell activating epitopes of SE in theconjugates precluding the conjugate from stimulating a tumoricidal Tcell response. The antibody-bound SE conjugates as large immunecomplexes are also readily taken up by reticuloendothelial cells andtherefore diverted from targeting the tumor.

[0662] To solve this problem, antibodies specific for the idiotypicregion-of anti-SE antibodies are prepared by methods given in Example57. The anti-idiotype is preferably of the Ab2β reticuloendothelialcells which is an internal image anti-idiotype and has the capacity tomimic the antigen used to generate the SE-specific antibody. The Ab2γantibody recognizes an Id within the antigen binding site, acharacteristic similar to Ab2β but fails to exhibit biological mimicryof the antigen. This particular subset of anti-idiotype antibodies wouldbe the most desirable in the present case as it would not furtheractivate the host anti-SE response, however the others could be usefulas well. The anti-Ids could also be prepared in hybridoma or geneticform and used in vivo (via transfection of autologous antibody producingcells or implantable chambers) to provide a continuous amount of anti-idfor the duration of use of the SE-conjugates).

[0663] The anti-Ids are prepared and administered as less immunogenicFab, F(ab)′₂, or Fv fragments and humanized to avoid alloimmunization.In the case of SEB, the dominant B cell epitope is the C-terminal region(aa 225-234) which may be used to isolate the major SE specificantibodies. Anti-SE idiotypic antibodies are detected and characterizedas given in Example 57. The amount of anti-idiotype antibodyadministered is sufficient to neutralize all the circulating SE specificantibodies. Doses would range from 100 ng to 1000 mg and could bepredicted based on in vitro neutralization tests determining the amountof antibody required to bind all of the antigen in a small volume ofserum. When the level of these antibodies is undetectable, the SE-tumorspecific antibody conjugate is administered in doses effective to killthe tumor. This could range from 2-15 ng/kg-100 ug/kg of SE in theconjugate. Devoid of SE specific antibodies to neutralize T cellactivating activity and divert the SE conjugate from the tumor site, theSE conjugate is able to target the tumor in vivo and initiate atumoricidal response. The preferred route of injection is intravenousbut other parenteral routes such as intraperitoneal, intrathecal andintratumoral may be useful as well. The preferred method ofadministration is via infusion although injection, sustained releaseformulations and microinfusion or osmotic pumps may also be useful.

[0664] Epitopes that Saturate MHC Class II and Neutralize SE AntibodyBinding.

[0665] The present invention contemplates the use of superantigenpeptide or nucleotide epitopes recognized by superantigen specificantibodies in the circulation of cancer patients which neutralize thetherapeutic effect of superantigen specific antibodies. Followingneutralization, therapeutic superantigen-tumor specific ligandconjugates are administered which target the tumor and express theirtumoricidal effects in vivo. A dominant epitope on SEB recognized byanti-SEB antibodies is the sequence 225-234 (Nishi et al., J. Immunol.158: 247-254 (1997) and an epitope on SEA recognized by anti-SEAantibodies is the sequence 121-149 (Hobieka et al., Biochem. Biophys.Res. Comm. 223: 565-571 (1996).

[0666] A preferred epitope to neutralize SE-specific antibodies also hasmarginal superantigen properties compared to the native agent as shownby their inability to induce proliferation of T cells via Vβ and expressmRNA encoding TH1 cytokines IL-2, and IFNγ. An example of an SEB mutantwith minimal T cell proliferative activity and cytokine activation has asingle residue change from the wild type around a conserved leucine(L45). These mutants are designated Q43P, F44P or L45R (Woody M A etal., J. Infect. Dis. 177:1013-1022 (1998). An anti-idiotype peptide orwhole antibody, F(ab′)₂ or Fv, specific for the variable region of theSE antibody is also useful in saturating the antigen binding region ofthe SE antibody. The latter peptide is prepared as a monomer, dimer orpolymer or attached to a carrier conjugates.

[0667] A second peptide or antibody (including F(ab′)₂ or Fv) specificfor the alpha1 subunit of the DR class II molecules (the MHC class IIbinding site for SEB) blocks SEB binding to its non-polymorphic MHCclass II receptor and inhibits class II-TCR interactions. This blockingconstruct saturates MHC class II binding sites so that when therapeuticSAg-tumor specific ligand conjugates are administered, they are notdiverted to MHC class II receptors. Further, the blocking construct doesnot interfere with the T cell activating function of the therapeuticSAg-mAb drug because the latter binds to tumor via its tumor specificantibodies and activates the T cell TCR via a non-MHC class II dependentmechanism.

[0668] The SE antibody neutralizing agent (either the SE antibodyneutralizing peptide or anti-idiotype antibody) and MHC class IIblocking agent (either inactive SE or anti-DR alpha 1antibody or inertSE peptide) are administered one hour before the administration of theSE-monoclonal antibody therapeutic conjugate. The amount of epitope oranti-idiotype antibody required to neutralize the anti-SE antibody isdetermined by measuring the amount of SE antibody present in the serumof the subject. A therapeutic dose is selected that neutralizes all ofthe anti-SE binding activity in the serum. The MHC class II bindingpeptide or anti-MHC class II alpha1 domain antibody is administered inamounts sufficient to saturate all the MHC class II alpha I domainreceptors. One hour after administration of the two blocking constructs,the therapeutic SAg-mAb conjugate is used to localize and target tumorin vivo. Preferentially, the tumor specific antibody or receptor ligandin the conjugate has a greater affinity for the tumor than the SAg inthe conjugate has for the class II molecule thus preventing the SAg frombinding all MHC class II receptors and favoring binding of the conjugateto the tumor.

[0669] To reduce affinity of the SE or SPE-mAb conjugate for endogenousMHC class II binding sites, the high affinity Zn⁺⁺-dependent MHC classII binding sites in SEA, SEC2, SEC3, SED, SPEA, SPEC, SPEG, SPEH, SMEZ,SMEZ2, Mycoplasma arthritides are deleted or replaced by inertsequence(s) or amino acid(s). These structural alterations in SE or SPEAreduce the affinity for MHC class II receptors from a K_(d) of 10⁻⁷ or10⁻⁸ to 10⁻⁵. In the case of SEB and SSA and other SEs or SPEs whichhave multiple low affinity MHC class II binding sites (K_(d) 10⁻⁵-10⁻⁷),alteration of the MHC class II binding sites is not necessary to furtherreduce affinity for MHC class II receptors; at the very least only oneor two of these sites near the N terminus of the molecule may requirestructural deletion of substation.

[0670] To further reduce affinity of the SE or SPE-mAb conjugate forendogenous (and sometimes preexisting) SE specific antibodies, the SEdominant epitope on the SE molecule in the conjugate is either removedor replaced by an inert sequence. For example, the dominant B cellepitope in SEB, amino acids 225-234 is replaced by sequence 209-212 fromSPEA.

[0671] Tumor specific antibodies, Fab, F(ab)′2 or single chain Fab or Fvfragments conjugated to SE or SPE in the SE or SPEA-mAb conjugate areused which have a higher affinity for tumor antigens (K_(d) 10⁻¹¹-10⁻¹⁴or lower) than the SE or SPE class II binding sites and SE epitopes(dominant or primary, secondary or tertiary etc.) in the conjugate havefor endogenous MHC class II receptors (K_(d) 10⁻⁵ to 10⁻⁷) andcirculating SE antibodies (K_(d) 10⁻⁷ to 10⁻¹¹) respectively.

[0672] To further enhance the affinity of the tumor specific antibody inthe conjugate for tumor cells in vivo, the tumor specific antibodies areused which are specific for more than one antigenic structures on thetumor, tumor stroma or tumor vasculature or any combination thereof. Thetumor specific antibody or Fab′2, Fab or single chain Fv fragments aremono or divalent like IgG, polyvalent for maximal affinity like IgM orchimeric with multiple tumor (tumor stroma or tumor vasculature)specificities. Thus, when the SE or SPE-mAb conjugate is administered invivo, it will preferentially bind to tumor cells rather than toendogenous SE antibodies or MHC class II receptors.

[0673] The SE-mAb or SE-monoclonal Fab/Fv conjugates described above areadministered parenterally, intratumorally, intrathecally,intraperitoneally, intrapleurally by infusion or injection inconventional or sustained release vehicles as given in Section 66 indosages of 0.01 ng/kg to 100 μg/kg using protocols given in Examples 5,7, 14, 15, 16, 18-23, 38.

60. New Streptococcal Pyrogenic Exotoxins, Staphylococcal Enterotoxinsand SETs for Tumor Therapy

[0674]Streptococcal pyrogenic exotoxins SPEA, SPEB, SPEC, SPEG, SPEH,SPEH SME-Z, SME-Z₂ and SSA are superantigens induce tumoricidal effects.SPEG, SPEH, and SPEJ genes were identified from the Streptococcuspyogenes M1 genomic database at the University of Oklahoma. A fourthnovel gene (smez-2) was isolated from the S. pyogenes strain 2035, basedon sequence homology to the streptococcal mitogenic exotoxin z (smez)gene. SMEZ-2, SPE-G, and SPE-J are most closely related to SMEZ andstreptococcal pyrogenic exotoxin (SPE)-C, whereas SPE-H is most similarto the staphylococcal toxins than to any other streptococcal toxin.Recombinant (r)SMEZ, rSMEZ-2, rSPE-G, and rSPE-H were mitogenic forhuman peripheral blood lymphocytes. SMEZ-2 is the most potentsuperantigen (SAg) discovered thus far. All toxins, except rSPE-G, wereactive on murine T cells, but with reduced potency. Binding to a humanB-lymphoblastoid line was shown to be zinc dependent with high bindingaffinity of 15-65 nM. Evidence from modeled protein structures andcompetitive binding experiments suggest that high affinity binding ofeach toxin is to the major histocompatibility complex class II β chain.Competition for binding between toxins was varied and revealedoverlapping but discrete binding to subsets of class II molecules in thehierarchical order (SMEZ, SPE-C)>SMEZ-2>SPE-H>SPE-G. The most commontargets for these SAgs were human Vβ2.1- and Vβ4-expressing T cells.

[0675] There are four naturally occurring SPEA alleles, and three ofthese, SPEA1, SPEA2, and SPEA3, encode toxins differing by a singleamino acid. The toxin encoded by SPEA4 is 9% divergent from the otherthree, with 26 amino acid changes. Twenty mutant forms of SPEA1 (SPEAencoded by allele 1), and the mutant toxins were analyzed for mitogenicstimulation of human peripheral blood mononuclear cells, affinity forclass II major histocompatibility complex molecules (DQ), and disulfidebond formation. Residues necessary for each of these functions wereIdentified., The product of allele 2, SPEA2, had slightly higheraffinity for the class II MHC molecule compared with SPEA1 but notsignificantly greater mitogenic activity. SPEA3, however, wassignificantly increased in mitogenic activity and affinity for class IIMHC compared with SPEA1. Thus, there is evidence that the toxin encodedby some of the highly virulent S. pyogenes STSS-associated isolates is amore active form of SPEA.

[0676] A new ˜28-kDa superantigen protein designated streptococcalsuperantigen (SSA), was isolated from culture supernatants. SSAstimulated proliferation of human T cells bearing Vβ1, Vβ3, Vβ5.2, andVβ15 in an MHC class II-dependent manner. N-terminal sequencing foundthe first 24 residues of SSA to be 62.5% identical to staphylococcalsuperantigens SEB, SEC1, and SEC3.

[0677] Newer Staphylococcal enterotoxins SEG, SEH, SEI, SEJ, SEK, SEL,SEM, ENT, ENT1, ENT2, also show superantigenic activity and are capableof inducing tumoricidal effects. The homology of these toxins to othertoxins in the family ranges from 27-64%. Selective expansion of TCR Vβsubsets has been demonstrated for each. (Jarraud S et al., J. Immunol.166: 669-677 (2001); Jarraud S et al., J. Clin. Microbiol. 37: 2446-2449(1999); Munson, S H et al., Infect. Immun. 66:3337-3345 (1998 The abovestreptococcal and newer enterotoxins retain T cell activation and VBusage. SPEA, SPEC, SPEG, SPEH, SME-Z, SME-Z₂, SEG, SEH, are known toutilize zinc as part of high affinity MHC class II receptor. Amino acidsubstitution(s) at the high affinity zinc dependent class II bindingsite is used to reduce their affinity for MHC class II receptor. Tumorlocalization is insured by the fusion with tumor specific antibodies,F(ab′)₂ or single chain Fv fragments. Additional or alternative tumorlocalizing motifs may be added to the toxin molecules which include butare not limited to an RGD motif, VEGF (localizing to KDR tyrosine kinasereceptors on vascular endothelium) and other tumor receptor ligands.

[0678] These proteins and their homologues are isolated andcharacterized as in Example 62 and Section 64. These proteins and theirhomologues are useful as preventative or therapeutic antitumor vaccinesaccording to Examples 4, 15,16, 18-23 and in nucleic acid form as inExample 31. They are administered as given in Example 14 preferablyparenterally by injection or infusion. They are also useful ex vivo forproducing a population of tumor specific effector cells (T cells, γ/δTcells NKT cells, NK cells) for adoptive immunotherapy of cancer(Examples 2-5, 7,14, 15, 16, 18-23, 68).

[0679] In the present invention, these agents in protein or nucleic acidform is used to treat tumors. These agents may be administeredintratumorally or parenterally by injection or infusion as given inExamples 14-16, 18-23. Allelic forms, homologues or mutants of thenative agents as given in section 65 are also useful in treatment. Theyare used to induce tumor killing as a vaccine or against establishedtumors under protocols given in Examples 14-16, 18-23

62. Staphylococcal Exotoxin Like Proteins

[0680] The Staphylococcal exotoxin-like proteins (SETs) encoded by a newgroup of S. aureus exotoxin genes with sequence homology to the nativeenterotoxins and retain the consensus sequences that are found instaphylococcal and streptococcal exotoxins and toxic shock syndrometoxin 1. These agents stimulate the production of interleukin-1β, IL-6,and TNFα by human peripheral blood mononuclear cells and inducetumoricidal responses (Williams, R J et al., Infect. Immun. 68:4407-4415(2000)). Natural preexisting antibodies specific for these agents arenot generally present in human serum.

[0681] The four complete set genes encode proteins of between 227 and234 amino acids, which have 38 to 53% homology to each other. Thepartial set2 gene encodes the C-terminal 172 amino acids of thisprotein. An alignment of the amino acid sequences of the SET proteins isshown in FIG. 2 of Williams et al., supra. Each of the set proteins(apart from set2, for which the N-terminal region has not beensequenced) possesses putative N-terminal signal sequences, as determinedusing the Signal algorithm for gram-positive bacteria, indicating thatthese are secreted proteins. The mature extracellular proteins arepredicted to consist of 201 (set1), 204 (set3), 202 (set4), and 207(set5) amino acids. Although the similarity between the set proteins andthe known staphylococcal and streptococcal exotoxins is low, beingaround 25%, each of the set proteins possesses thestaphylococcal/streptococcal exotoxin consensus signature 2(K—X2-[LIV]-X2-[LIV]-D-X3-R—X2-L-Xs-[LIV]-Y) as determined by searchesof the PROSITE database. In addition, the set proteins have an aminoacid sequence which is similar to the exotoxin consensus signature 1(PRO-SITE, PCOC00250). set1, set2, and set5 have the greatest homologyto SPEC and 25% sequence identity, respectively), while set3 and set4are most closely related to TSST-1 (25 and 27% identity, respectively).Based on this homology, the set sequences were modeled against twotarget protein structures, TSST-1 (PDB file 2tss) and SPEC (PDB fileIan8). The protein models of set1 and set5 depict two domain structuresjoined by a central α-helix. Domain B has topology equivalent to theoligosaccharide- and oligonucleotide-binding fold (OB-fold) found inSPEC and other unrelated toxins. However, the A domain of set1 and set5differs from that of SPEC in that it has one less β-strand in theβ-grasp motif. The missing β-strand corresponds to β12 in the SPEC modeland is important in zinc binding and in providing biological activity.The protein models of set5 and set4 also depict a two-domain structurejoined by a central α helix (Williams et al., supra). Once again, the Bdomain forms a β-barrel structure. The β-grasp motif of the A domain inset3 and set4 is similar to that of TSST-1. However, the shortN-terminal α-helix (aj of TSST-1 is not maintained in set3 and set4.Although there is low amino acid sequence identity between the setproteins and those of the target protein structures, these theoreticalmodels demonstrate that the set proteins could adopt typicalsuperantigen-type structures.

[0682] These proteins and their homologues are isolated andcharacterized as in Example 63 and Section 65. These proteins and theirhomologues are useful as preventative or therapeutic antitumor vaccinesaccording to Examples 4, 15,16, 18-23 and in nucleic acid form as inExample 31. They are administered as given in Example 14 preferablyparenterally by injection or infusion. They are also useful ex vivo forproducing a population tumor specific effector cells (T cells, γ/δ Tcells, NKT cells, NK cells) for adoptive immunotherapy of cancer(Examples 2-5, 7,14, 15, 16, 18-23, 68).

[0683] These agents may be administered intratumorally or parenterallyby injection or infusion as given in section 14-16, 18-23. Allelicforms, homologues or mutants of the native agents as given in section 65are also useful in treatment. They are used to induce tumor killing as avaccine or against established tumor under protocols given in Examples14-16, 18-23.

62. Streptococcal Pyrogenic Exotoxin B with RGD sites, CysteinylProtease (Interleukin 1β Convertase) and Metalloproteinase Activity

[0684] The structural gene for SPE B was cloned and characterized. ThespeB gene (1194 bp) encodes a 398-amino-acid protein. A presumed signalpeptide of 26 amino acids is cleaved to result in a mature extracellularprotein (zymogen) with 372 residues. Subsequently, Kapur et al. (109)used an automated DNA sequencing strategy to study speB allelicvariation in 67 strains expressing 39 M protein serotypes and fiveprovisional M serological types and representing 50 phylogeneticallydistinct clones identified by multilocus enzyme electrophoresis. ThespeB gene is well conserved and allelic variation is due solely toaccumulation of point mutations. A total of 39 distinct speB alleles wasidentified, and based on predicted amino acid sequences, 33 of the 39alleles would encode one of three mature protease variants that differfrom one another at only one or two amino acids clustered in a10-amino-acid region (residues 308-317) that contains a linear B-cellepitope. A total of 61 of 64 (95%) strains examined, including strainsrepresenting all 39 alleles sequenced, expressed a product that reactedwith polyclonal rabbit antisera specific for purified cysteine protease.The data demonstrated that the zymogen and protease are very wellconserved, and virtually all strains expressed the speB gene under theconditions assayed.

[0685] The human pathogenic bacterium Streptococcus pyogenes (group Astreptococcus) produces a cysteine protease that is thought to be animportant virulence factor. The protein is made as an inactive zymogenof 40,000 Da, which is converted to a 28,000-Da active form byproteolysis or auto-catalytic truncation in reducing conditions of theN-terminal 118 amino acid residues (the prosegment), to generate themature, active protease (mSpeB) of 253 residues (27.6 kDa). Thecatalytic site differs from most other cysteine proteases in that itlacks the Asn residue of the Cys-His-Asn triad. The prosegment has aunique fold and inactivation mechanism that involves displacement of thecatalytically essential His residue by a loop inserted into the activesite. The structure also reveals the surface location of anintegrin-binding Arg-Gly-Asp (RGD) motif that is a feature unique toSpeB among cysteine proteases.

[0686] Virtually all S. pyogenes strains have the structural gene (speB)and express active enzyme The zymogen or active protease is also knownas streptococcal pyrogenic exotoxin B (SPE B). Sequence analysis of the1,197-bp speB gene from 67 isolates shows that the gene is highlyconserved in natural populations of S. pyogenes. The protease cleavesfibronectin (FN) and vitronectin, two abundant extracellular matrixproteins involved in maintaining host tissue integrity and alsoactivates a 66 kilodalton human endothelial cell matrix metalloprotease.In addition, the protease cleaves human interleukin-1β precursor togenerate mature interleukin-1β with full biologic activity. PurifiedSpeB causes a cytopathic effect on cultured human endothelial cells.Biochemical and genetic analyses have shown that both zymogen and activeenzyme contain only one cysteine residue (C-192) which participates inenzyme active-site formation and catalysis.

[0687] All serotype M1 organisms, which are the most common cause ofinvasive disease, and some 20% of other isolates, have an RGD sequencemotif at this site. RGD motifs mediate ligand binding to many integrinsand the RGD-containing variant of SpeB binds to the human integrinsα_(v)β₃ and α_(q1b)β₃ through this motif (Kagawa et al., Proc. Nat'lAcad. Sci. USA 97: 2235-2240 (2000)). Integrins are heterodimericmembrane proteins located on the surface of mammalian cells which areoverexpressed in invasive tumors. The 3-aa sequence Arg-Gly-Asp (RGD) iscritical for recognition by many integrins. SPEB as well as microbialpathogens as Borrelia burgdoferi, Yersinia spp., Bordetella pertussis,adenovirus, echovirus, foot-and-mouth disease virus, and hantavirusesbind integrins usually through an RGD motif.

[0688] In the SpeB structure, Gly-163 and Asp-164 are fullysolvent-exposed. The exposure of the Asp-164 side chain is particularlysignificant because integrin binding is believed to occur throughcoordination of the Asp residue of the RGD motif to a divalent metal onthe integrin receptor. The RGD motif in SpeB is located on a surfaceloop inserted between a short β-strand and the α-helix that leads intothe C-terminal protease domain and is remote from both the active siteof mSpeB and the site where the prosegment associates with the protease,implying that integrin binding through this RGD motif can occur for boththe zymogen and mature protease forms of SpeB. Hence, when the proteaseis bound through its RGD motif, its active site should be fully exposed.Integrin binding thereby could concentrate mSpeB at the host cellsurface, enhancing the efficiency of local degradation of host moleculessuch as fibronectin and vitronectin.

[0689] These proteins and their homologues are isolated andcharacterized as in Example 64 and Section 65. These proteins and theirhomologues are useful as preventative or therapeutic antitumor vaccinesaccording to Examples 4, 15,16, 18-23 and in nucleic acid form as inExample 31. They are administered as given in Example 14 preferablyparenterally by injection or infusion. They are also useful ex vivo forproducing a population of tumor specific effector cells (T cells, γ/δTcells NKT cells, NK cells) for adoptive immunotherapy of cancer(Examples 2-5, 7,14, 15, 16, 18-23, 68).

[0690] In the present invention, SPEB in protein or nucleic acid form isused to treat tumors.

[0691] These agents may be administered intratumorally or parenterallyby injection or infusion as given in section 14-16, 18-23. Allelicforms, homologues or mutants of the native agents as given in section 65are also useful in treatment. They are used to induce tumor killing as avaccine or against established tumors under protocols given in Examples14-16, 18-23.

63. Non-Staphylococcal Enterotoxins and Streptococcal PyrogenicExotoxins as Tumoricidal Agents: Yersinia Species Superantigen with aNatural RGD Motif as a Tumoricidal Agent

[0692]Yersinia pseudotuberculosis has a molecular weight 14,500 and hasno C—C loop. the DNA expressed in Escherichia coli DH10B. Sequencing ofthe YPM gene revealed that the gene contained an open reading frame of456 base pairs encoding a precursor form of 151 amino acid residues withm.w. 16,679 that is processed into a mature form of 131 amino acidresidues with m.w. 14,529. Homology analysis revealed that the homologyof amino acid sequence is quite low among YPM and other well knownbacterial superantigens. It is a known superantigen with the ability tobind directly to MHC class II molecules and selectively stimulate T cellpopulations bearing particular Vβ elements in TCR The recombinant YPMexpressed by the cloned gene exerted a mitogenic activity on human PBMCat a concentration of approximately 1 pg/ml. with VB usage 3, 9, 13.1-3in humans and in mice 7, 8.1-3. Like the native YPM, the cloned YPMrequired the expression of MHC class II molecules on accessory cells inthe induction of IL-2 production by human T cells (Tohru M et al., J.Immunol. 154: 5228-5234 (1995); Ito, Y et al., J. Immunol. 154:5896-5906 (1995); Uchiyama T et. al., J. Immunol. 151: 4407 (1993)). Theyersinia superantigen also contains an RGD motif hence, this molecule innative form has tumor localizing capability. In addition, unlike some ofthe SEs or SPEs, most humans do not have preexisting antibodies to YPM.

[0693] These proteins and their homologues are isolated andcharacterized as in Example 65 and Section 65. These proteins and theirhomologues are useful as preventative or therapeutic antitumor vaccinesaccording to Examples 4, 15,16, 18-23 and in nucleic acid form as inExample 31. They are administered as given in Example 14 preferablyparenterally by injection or infusion. They are also useful ex vivo forproducing a population of tumor specific effector cells (T cells, γ/δTcells, NKT cells, NK cells) for adoptive immunotherapy of cancer(Examples 2-5, 7,14, 15, 16, 18-23, 68).

[0694] These agents may be administered intratumorally or parenterallyby injection or infusion as given in section 14-16, 18-23. Allelicforms, homologues or mutants of the native agents as given in section 65are also useful in treatment. They are used to induce tumor killing as avaccine or against established tumor under protocols given in Examples14-16, 18-23.

63. Viral Vectors Expressing Antibodies or Ligands for Tumor Structuresor Receptors and DNA Binding Proteins e.g., Protamine, Poly-L-LysineBinding Superantigen DNA

[0695] To circumvent the problem of naturally occurring antibodies toSEs, the present invention contemplates the use of nucleic acidsencoding the SEs which are transported to the tumor by tumor localizingmotifs expressed on the construct. The superantigen nucleic acids arecarried as part of a viral vector or connected to the vector by a DNAbinding protein such as protamine or poly L-lysine. The presentinvention contemplates viral vectors such as adenoviruses which expresstumor targeting motifs including but not limited to intact antibodies orfragments of antibodies in any form e.g., single chain Fv or F(ab)′2fragments. These antibodies are preferably of high affinity for atargets on tumor cells which include but are not limited to the Erb/Neureceptor and tumor specific antigens. Vectors also carry nucleic acidsencoding tumor localizing motifs which are expressed on their surfaceincluding but not limited to RGD motifs which bind to integrins α_(v)β₃and α_(q1b)β₃ in tumors, chemokine receptors e.g., CXCR4 which bind toCXCL12.

[0696] Vectors, which themselves have natural and intrinsic RGD bindingmotifs including but not limited to adenovirus, echovirus, foot andmouth disease virus and Hanta virus, are used. The disease causingsegment(s) of the these viruses are attenuated or deleted while theirtumor localizing properties are retained.

[0697] DNA binding proteins which bind the virus to exogenoussuperantigen DNA include but are not limited to protamine orpoly-l-lysine. Nucleic acids encoding these DNA binding proteins areintegrated into the viral vector which are expressed on the viralsurface. Exogenous superantigen DNA binds to the DNA binding protein(e.g., protamine or poly-l-lysine) expressed on the viral surface.

[0698] A tumor localizing motif on the vector makes contact with thetumor cell receptor or antigen wherein the superantigen DNA (as eitheran integral part of the virus or exogenously bound to the virus) isendocytosed. Once inside the target tumor cell, the superantigen DNA istranscribed into a functional superantigen polypeptide which isexpressed in the tumor or secreted locally where it can exert itstumoricidal effects. All of the SEs, SPEs and SETs and non SEsuperantigens including but not limited to Yersinia pseudotuberculosis,Mycoplasma arthitides, rabies and Clostridia heat shock proteins may beused in this construct.

[0699] In an additional embodiment, nuceleic acids encodingsuperantigen-tumor specific antibody (Fab′)₂, Fv, Fd immunoconjugates inwhich the superantigen is fused to the Fc region of the tumor specificantibody or antibody fragment are transfected directly into tumor cells.The superantigens produce the immunoconjugates which can then localizeto surrounding tumor and induce a tumoricidal response. Methods forproduction of these immunoconjugates are given in Example 66.

[0700] Small synthetic peptide, [K]₁₆RGD, is a receptor-mediatedsuperantigen gene delivery vehicle. [K]₁₆RGD is a bifunctional linearpeptide that comprises an integrin-targeting RGD component and asuperantigen DNA-binding moiety consisting of 16 lysine residues. Thepeptide binds to cell surface integrins, forms distinct, protectivecomplexes with superantigen DNA, and can deliver superantigen DNA tocells for transgene expression. The level of superantigen geneexpression obtained is dependent on the [K]₁₆RGD-to-DNA ratio and isenhanced in the presence of the endosomal buffer chloroquine orlysosomal disrupting agents during gene delivery. Levels of reportergene expression obtained under ideal conditions with [K]_(|6)RGDapproach or even exceed those obtained with DNA delivered byLipofectamine. See Example 66.

[0701] These proteins and nucleic acids and their homologues areisolated and characterized as in Example 66 and Section 65. Theseproteins and their homologues are useful as preventative or therapeuticantitumor vaccines according to Examples 4, 15,16, 18-23 and in nucleicacid form as in Example 31. They are administered as given in Example 14preferably parenterally by injection or infusion. They are also usefulex vivo for producing a population of tumor specific effector cells (Tcells, γ/δT cells, NKT cells, NK cells) for adoptive immunotherapy ofcancer (Examples 2-5, 7,14, 15, 16, 18-23, 68).

[0702] In the present invention, these agents in protein or nucleic acidform are used to treat tumors. These agents may be administeredintratumorally or parenterally by injection or infusion as given insection 14-16, 18-23, 66. Allelic forms, homologues or mutants of thenative agents as given in section 65 are also useful in treatment. Theyare used to induce tumor killing as a vaccine or against establishedtumors under protocols given in Examples 14-16, 18-23, 66.

64. Functional Homologues & Derivatives of Proteins of Peptides

[0703] All of the protein and nucleic acid compositions given herein areintended to encompass functional derivatives. All of the functionalderivatives of the fusion partners for superantigens described in thisapplication are encompassed by this invention. Similarly, Staphylococcalenterotoxins or superantigens are intended to encompass functionalderivatives of a particular superantigen or enterotoxin.

[0704] By “functional derivative” is meant a “fragment,” “variant,”“homologue,” “analogue,” “fusion protein,” or “chemical derivative”,which terms are defined below. A functional derivative retains at leasta portion of the function of the native protein monomer which permitsits utility in accordance with the present invention.

[0705] A “fragment” refers to any shorter peptide. A “variant” of refersto a molecule substantially similar to either the entire protein or apeptide fragment thereof. Variant peptides may be conveniently preparedby direct chemical synthesis of the variant peptide, using methodswell-known in the art.

[0706] All or the compositions given herein or claimed as part of a newinvention include the homologues of that composition. A homologue refersto a natural protein, encoded by a DNA molecule from the same or aprotein. Homologues, as used herein, typically share about 50% sequencesimilarity at the DNA level or about 18% sequence similarity in theamino acid sequence. Homologues are more aptly quantitated in thestatistical programs given below. An example a homologue of a nativestaphylococcal enterotoxin would be any structure including allsubstitution, deletion or addition mutants, derivatives, fusionproteins, chimeric proteins, fragments, conjugates, synthetic andnaturally occurring structures with a Z value >10 in the Lipman-PearsonFASTA/FASTP program.

[0707] An “analogue” refers to a non-natural molecule substantiallysimilar to either the entire molecule or a fragment thereof

[0708] A “chemical derivative” contains additional chemical moieties notnormally a part of the peptide. Covalent modifications of the peptideare included within the scope of this invention. Such modifications maybe introduced into the molecule by reacting targeted amino acid residuesof the peptide with an organic derivatizing agent that is capable ofreacting with selected side chains or terminal residues.

[0709] The recognition that the biologically active regions of theenterotoxins, for example, are substantially structurally homologousenables predicting the sequence of synthetic peptides which exhibitsimilar biological effects in accordance with this invention (Johnson,L. P. et al., Mol. Gen. Genet. 203:354-356 (1886).

[0710] A common method for evaluating sequence homology, and moreimportantly, for identifying statistically significant similarities ofthe proteins, peptides and nucleic acids given herein is by Monte Carloanalysis using an algorithm written by Lipman and Pearson to obtain a Zvalue (FASTA). According to this analysis, Z>6 indicates probablesignificance, and Z>10 is considered to be statistically significant(Pearson, W. R. et. al., Proc. Natl Acad Sci. USA, 85:2444-2448 (1988);Lipman, D. J. et al, Science 227:1435-1441 (1985)). Synthetic peptidescorresponding to the compositions and enterotoxins and all othermolecules described herein are characterized in that they aresubstantially homologous in amino acid sequence to an enterotoxin orother native molecule to which it is being compared with statisticallysignificant (Z>6) sequence homology and similarity to include alignmentof cysteine residues and similar hydropathy profiles.

[0711] The Lipman-Pearson FASTA program may be used to determinehomology of a given protein using the BLOSUM 50 or PAM 250 scoringmatrix, gap penalties of −12 and −2 and the PIR or SwissPROT database.The results are expressed as Z values or E ( ) values. For the presentdatabase (2001), the Z>13 indicates statistical significance.

[0712] Preferred Methods for Establishing Statistical Significance ofProteins or Nucleic Acids

[0713] The most widely used and preferred methodology determines thepercent identity of two amino acid sequences or of two nucleic acidsequences. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred method of alignment, Cys residues are aligned. In apreferred embodiment, the length of a sequence being compared is atleast 20%, preferably at least 40%, more preferably at least 50%, evenmore preferably at least 60%, and even more preferably at least 70%,80%, or 90% of the length of the reference sequence. The amino acidresidues (or nucleotides) at corresponding amino acid positions (ornucleotide) positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue (or nucleotide) asthe corresponding position in the second sequence, then the moleculesare identical at that position (as used herein amino acid or nucleicacid “identity” is equivalent to amino acid or nucleic acid “homology”).The percent identity between the two sequences is a function of thenumber of identical positions shared by the sequences, taking intoaccount the number of gaps, and the length of each gap, which need to beintroduced for optimal alignment of the two sequences.

[0714] The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. 48:444-453 (1970) algorithm which has been incorporated intothe GAP program in the GCG software package (available athttp://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1,2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (available athttp://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. Inanother embodiment, the percent identity between two amino acid ornucleotide sequences is determined using the algorithm of E. Meyers andW. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into theALIGN program (version 2.0), using a PAM120 weight residue table, a gaplength penalty of 12 and a gap penalty of 4.

[0715] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstpublic databases, for example, to identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul et al. (1990) J. Mol. Biol.215:403-10. BLAST nucleotide searches can be performed with the NBLASTprogram, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to human or murine nucleic acid molecules. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to human ormurine protein molecules of the invention. To obtain gapped alignmentsfor comparison purposes, Gapped BLAST can be utilized as described inAltschul et al. (1997) Nucleic Acids Res. 25:3389-3402. When utilizingBLAST and Gapped BLAST programs, the default parameters of therespective programs (e.g., XBLAST and NBLAST) can be used. Seehttp://www.ncbi.nlm.nih.gov.

[0716] Variants

[0717] One group of variants are those in which at least one amino acidresidue in the peptide molecule, and preferably, only one, has beenremoved and a different residue inserted in its place. For a detaileddescription of protein chemistry and structure, see Schulz, G. E.Principles of Protein Structure Springer-Verlag, New York, 1978, andCreighton, T. E.,

[0718]Proteins: Structure and Molecular Properties, W. H. Freeman & Co.,San Francisco, 1983, which are hereby incorporated by reference. Thetypes of substitutions which may be made in the protein or peptidemolecule of the present invention may be based on analysis of thefrequencies of amino acid changes between a homologous protein ofdifferent species, such as those presented in Table 1-2 of Schulz et al(supra) and FIG. 3-9 of Creighton (supra). Based on such an analysis,conservative substitutions are defined herein as exchanges within one ofthe following five groups:

[0719] 1. Small aliphatic, nonpolar or slightly polar residues: Ala,Ser, Thr (Pro, Gly);

[0720] 2. Polar, negatively charged residues and their amides: Asp, Asn,Glu, Gln;

[0721] 3. Polar, positively charged residues: His, kg, Lys;

[0722] 4. Large aliphatic, nonpolar residues: Met, Leu, Val (Cys); and

[0723] 5. Large aromatic residues: Phe, Tyr, Trp.

[0724] The three amino acid residues in parentheses above have specialroles in protein architecture. Gly is the only residue lacking any sidechain and thus imparts flexibility to the chain. Pro, because of itsunusual geometry, tightly constrains the chain. Cys can participate indisulfide bond formation which is important in protein folding. Tyr,because of its hydrogen bonding potential, has some kinship with Ser,Thr, etc.

[0725] Substantial changes in functional or immunological properties aremade by selecting substitutions that are less conservative, such asbetween, rather than within, the above five groups, which will differmore significantly in their effect on maintaining (a) the structure ofthe peptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain. Examplesof such substitutions are (a) substitution of gly and/or pro by anotheramino acid or deletion or insertion of Gly or Pro; (b) substitution of ahydrophilic residue, e.g., Ser or Thr, for (or by) a hydrophobicresidue, e.g., Leu, Phe, Val or Ala; (c) substitution of a Cys residuefor (or by) any other residue; (d) substitution of a residue having anelectropositive side chain, e.g., Lys, Arg or His, for (or by) a residuehaving an electronegative charge, e.g., Glu or Asp; or (e) substitutionof a residue having a bulky side chain, e.g., Phe, for (or by) a residuenot having such a side chain, e.g., Gly.

[0726] Most deletions and insertions, and substitutions according to thepresent invention are those which do not produce radical changes in thecharacteristics of the protein or peptide molecule. However, when it isdifficult to predict the exact effect of the substitution, deletion, orinsertion in advance of doing so, one skilled in the art will appreciatethat the effect will be evaluated by routine screening assays, forexample direct or competitive immunoassay or biological assay asdescribed herein. Modifications of such proteins or peptide propertiesas redox or thermal stability, hydrophobicity, susceptibility toproteolytic degradation or the tendency to aggregate with carriers orinto multimers are assayed by methods well known to the ordinarilyskilled artisan.

[0727] In the present invention, functional derivatives or homologues ofproteins, peptides, enterotoxins or other related toxins and nucleicacids including fusion proteins, mutants (deletion and addition types),variants, conjugates with other proteins including but not limited toantibodies, F(ab′)₂, Fv or Fd fragments, receptors or receptor ligands,synthetic polypeptides and nucleic acids characterized by substantialstructural homology to staphylococcal enterotoxin A, enterotoxin B,enterotoxin C, enterotoxin D, enterotoxin E, enterotoxin F (TSST-1) andStreptococcal pyrogenic exotoxins A-H as well as the newer enterotoxins(SEG, SEH, SEI, SEJ, SEK, SEL, SEM), SETs 1-5 and non-enterotoxinsuperantigens (e.g., Yersinia pseudotuberculosis superantigen,Mycoplasma arthitidis superantigen) with statistically significantsequence homology and similarity (e.g., Z>6 in the Lipman and Pearsonalgorithm in Monte Carlo analysis (FASTA program) or the preferredmethodology for determining sequence similarity and identity of proteinsand nucleic acid as given above in this section (e.g., ALIGN, NBLAST,XBLAST programs as described above) are included in the invention. Allof the superantigen conjugates to other polypeptides, peptides (e.g.,verotoxins, chemokine receptors, costimulants, invasins, viral antigens)given in this application are considered to be structural homologues areincluded in this invention as structural homologues if they show Zvalues >10 or additional statistical criteria for inclusion as given inthis section.

[0728] Chemical Derivatives

[0729] Covalent modifications of the monomeric or polymeric forms ofprotein or peptide fragments thereof, of enterotoxins or peptidefragments thereof, or both are included herein. Such modifications maybe introduced into the molecule by reacting targeted amino acid residuesof the protein or peptide with an organic derivatizing agent that iscapable of reacting with selected side chains or terminal residues. Thismay be accomplished before or after polymerization.

[0730] Cysteinyl residues most commonly are reacted with a-haloacetates(and corresponding amines), such as 2-chloroacetic acid orchloroacetamide, to give carboxymethyl or carboxyamidomethylderivatives. Cysteinyl residues also are derivatized by reaction withbromotrifluoroacetone, a-bromo-(5-imidozoyl)propionic acid, chloroacetylphosphate, Nalkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl2-pyridyl disulfide, p-chloromercuribenzoate,2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0731] Histidyl residues are derivatized by reaction withdiethylprocarbonate at pH 5.5-7.0 because this agent is relativelyspecific for the histidyl side chain. Para-bromophenacyl bromide also isuseful; the reaction is preferably performed in 0.1 M sodium cacodylateat pH 6.0.

[0732] Lysinyl and amino terminal residues are reacted with succinic orother carboxylic acid anhydrides. Derivatization with these agents hasthe effect of reversing the charge of the lysinyl residues. Othersuitable reagents for derivatizing a-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;0-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

[0733] Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pK of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

[0734] The specific modification of tyrosyl residues per se has beenstudied extensively, with particular interest in introducing spectrallabels into tyrosyl residues by reaction with aromatic diazoniumcompounds or tetranitromethane. Most commonly, N-acetylimidizol andtetranitromethane are used to form 0-acetyl tyrosyl species and 3-nitroderivatives, respectively.

[0735] Carboxyl side groups (aspartyl or glutamyl) are selectivelymodified by reaction with carbodiimides as noted above. Aspartyl andglutamyl residues are converted to asparaginyl and glutaminyl residuesby reaction with ammonium ions.

[0736] Glutaminyl and asparaginyl residues may be deamidated to thecorresponding glutamyl and aspartyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Either form ofthese residues falls within the scope of this invention.

[0737] Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the a-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MoleculeProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and, in some instances, amidationof the C-terminal carboxyl groups.

[0738] Such derivatized moieties may improve the solubility, absorption,biological half life, and the like. The moieties may alternativelyeliminate or attenuate any undesirable side effect of the protein andthe like. Moieties capable of mediating such effects are disclosed, forexample, in Remington's Pharmaceutical Sciences, 16th ed., MackPublishing Co., Easton, Pa. (1980).

65. Administration of Superantigens in Controlled Release Formulations

[0739] Superantigens compositions in form of polypeptides, peptides,homologues (defined in section 65), fusion proteins, conjugates,fragments, mutants, chimeric proteins, variants and/or nucleic acids orribonucleic acid are administered via implantable controlled releaseformulations and matrices. These formulations include but are notlimited to poly-(D-, L- or DL-lactic acid/polyglycolide) polymer,ethylene-vinyl acetate (EVAc) which is applicable to polypeptides ingeneral—bioerodible polyanhydrides, polyimino carbonate, sodium alginatemicrospheres, hydrogels, D and poly DL (lactide/glycolide) copolymers,ethylene-vinyl acetate (EVAc: Elvax 40W, Dupont) among others which aregiven in great detail in the foregoing patents and publications whichare hereby incorporated by reference (U.S. Pat. Nos. 4,891,225,4,942,035, 4,877,606, 4,906474, 4,806,621, 4,789,516, 4,925,677, EP92918B1, EP No. EPO166596B1; Jeyanthi, R et al., J. Controlled Release,13:91-98 (1990); Greig, N et al. J. Controlled Release 11:61-78 (1990);Kaitsu, I. et al., J. Controlled Release 6: 249-263 (1987); Yang, M. B.et al., Cancer Res 49:5103-5107 (1989); Eckenhoff, B et al.,Biomaterials 2:89 (1981)).

[0740] Preferably the controlled release preparations are administeredin close proximity or actually into a administration. However tumor atother sites may also be treated in this way.

[0741] Superantigens and SETs of any kind including but not limited tostaphylococcal enterotoxins A, B, C, D, E, F, G H, I, J, K, L, M,streptococcal pyrogenic exotoxins, Y. pseudotuberculosis superantigen,M. arthitidis, C. perfringens exotoxin are prepared as in U.S. Pat. No.6,126,945, which is hereby incorporated by reference, Examples 62-65,71. They are also administered in native form or as biologically activefragments peptides, homologues (defined in section 65), fusion proteins,mutants, chimeric proteins, variants and/or nucleic acids or ribonucleicacids are administered parenterally and intratumorally, intrathecally,intraperitoneally, intrapleurally directly into tumor or to a site inclose proximity to the tumor using conventional soluble diluents andvehicles such as normal saline as given below in this section.

[0742] Dosages of superantigens, conjugates and homologues used foradministration of these compositions range from 5 ng to 500 microgramsonce every 7-10 or 30 days. They may be given as a single or multipledoses. For intraperitoneal and intrapleural administration, thesustained release superantigens are generally (but not always)administered after partial or complete removal of the ascites or pleuralfluid from these cavities by thoracentesis or paracentesis.

66. Sickled Erythrocytes as Carriers of Tumoricidal Agents

[0743] Sickled erythrocytes are known to be more adherent tomicrovascular endothelium than normal erythrocytes and to adhere to agreater extent under conditions of local hypoxia and acidosis. Theprimary pathologic defect in sickle cell disease is the abnormaltendency of hemoglobin S to polymerize under hypoxic conditions. Thepolymerization of deoxygenated hemoglobin S results in a distortion ofthe shape of the red cell and marked decrease in its deformability.These rigid cells are responsible for the vaso-occlusive phenomena whichare the hallmark of the disease.

[0744] Sickle red cells adhere to the microvascular endothelium for thefollowing reasons: Sickled cells have abnormally increased expression ofα₄β₁ integrin and CD36. Activation of platelets releases thrombospondin,which act as a bridging molecule by binding to a surface molecule, CD36,on an endothelial cell and to CD36 or sulfated glycans on a sicklereticulocyte. Inflammatory cytokines induce the expression ofvascular-cell adhesion molecule 1 (VCAM-1) on endothelial cells. Thisadhesive molecule binds directly to the α₄β₁ integrin on the sicklereticulocyte.

[0745] In the oxygenated state, the extent of sickle cell adhesion isdensity-class dependent: reticulocytes and young discocytes (SS1)greater than discocytes (SS2) greater than irreversible sickle cells andunsicklable dense discocytes (SS4). Hypoxemic conditions have no effecton adherence of normal erythrocytes but sickle erythrocyte adherence toendothelial cells is increased significantly. The least dense sickleerythrocytes containing CD36 and VLA-4+ expressing reticulocytes areespecially involved in hypoxia sensitive adherence. Selective secondarytrapping of SS4 (dense cells) occurs in post capillary venules wheredeformable SS cells are preferentially adherent. Vaso-occlusion isinduced by a combination of precapillarly obstruction, adhesion in postcapillary venules, and secondary trapping of dense erythrocytes. Thisinduces local hypoxia leading to increased polymerization of hemoglobinS and rigidity of SS erythrocytes. In this way the obstruction ismultiplied and extended to nearby vessels.

[0746] In the present invention, sickled erythrocytes are used to carrytumoricidal agents into the microvasculature of tumors. Sickle celltrait cells are preferred since they are normal under physiologicconditions but sickle and become adhesive in the acidotic and/orhypoxemic tumor microvasculature. Tumoricidal agents introduced into andcarried by sickled erythrocytes include oncolytic viruses including butnot limited to herpes simplex, adenoviruses, vaccinia, Newcastle Diseasevirus, autonomous parvoviruses, In addition, the adenovirus encodingthymidine kinase is transfected into tumor cells that are thensusceptible to lysis ganciclovir. Various oncolytic and tumor specificviruses with tumor specificity used to transfect sickle cells aredescribed in Kirn, D. et al., Nat. Med. 7:781-7 (2001).

[0747] In addition the sickled erythrocyte carry nucleic acids encodingtumoricidal agents including but not limited to C. perfringens exotoxin,pertussis toxin, verotoxins, pseudomonas exotoxins and superantigens,perforin, granzyme B, complement components (membrane attack complex),oxidized LDL, tumor specific antibodies alone or fused to toxinsincluding but not limited to superantigens, Pseudomonas exotoxins,ricin, clostridia toxin. The nucleic acid encodes a hemolysin such asbut not limited to E. coli hemolysin or staphylococcal alpha hemolysin.The sickled cell can also contain anaerobic bacterial spores such asclostridia species which can grow selectively in hypoxemic tissues. Thesickled erythrocyte also carries phage displays, exosomes, sickle cellvesicles, sec vesicles expressing tumor toxins or superantigens. Thetoxins may be fusion proteins of toxins with ligands: expressed on tumorvasculature or tumor such a EGF, inactivated factor VIII or antibodiesspecific for a wide variety of tumor antigens well known in the art.

[0748] The nucleic acids encoding these toxins and oncolytic and tumorspecific viruses are placed under the promoter of the heat sensitiveglobal operator (Example 69). When entering the hypoxic tumor, sicklederythrocyte adhere to the tumor vasculature. In the hypoxemicenvironment of the tumor, the hypoxia sensitive global promoter isactivated and induces the production lytic viruses and toxins. Sickledcells are disrupted and lyse releasing lytic virus and toxin into thehypoxic tumor. As the tumor site becomes more hypoxic, VCAM-1 andp-selectin expression on tumor endothelium are upregulated trapping morecirculating sickled cells in the tumor microcirculation to undergo lysiswith release of tumoricidal products into the tumor area.

[0749] The sickled cell is transfected preferably with the oncolyticviruses and toxins given above at a stage preferably before it isenucleated (Examples 1, 60, 69). Nucleated sickle reticulocytes are thepreferred cell for transfection although enucleated sickled cells willalso work (Example 69) Anaerobic bacterial spores such clostridia aretransfected into the sickled erythrocytes by endocytosis orelectroporation (Schrier S. Methods in Enzymology 149: 261-271 (1987);Tsong T Y Methods in Enzymology 149-259 (1987)). They are alsointroduced into sickle erythrocytes that have been lysed under hypotonicconditions and the membranes annealed with encapsulation of theanaerobic spores(Example 69).

[0750] Erythrocytes from subjects with sickle trait are preferredbecause these red cells are functionally and structurally normal in thecirculation but are activated to sickle in the hypoxic tumorvasculature. Here they assume the sickled configuration, adhere to theendothelium of the tumor microcirculation and obstruct microvasculaturein a manner similar to the homozygous SS erythrocytes.

[0751] The sickled erythrocytes are administered parenterally byinjection or infusion in a therapeutically effective amount of cells.This encompasses a volume of 1-25 cc of packed cells administered i.v.over a one hour period. These cells are used in protocols given inExample 14-16, 18-23, 66.

67. Transfection of Tumor Cells with Superantigen and Nucleic acidsEncoding the OX-40 Ligand or 4-1BB1 Ligand or Antibody (F(ab′)₂ or FvFragments) Specific for OX-40 Receptor or the 4-1BB1 Receptor

[0752] Two primary vaccine strategies have been used to boost Tcell-specific tumor immunity in animal models as well as in humanclinical trials. The first involves genetic modification of tumor cellsin vitro (transfection of immune-enhancing genes) and immunization withthe altered tumor cells in vivo. The second strategy involves expansionof tumor-reactive T cells either by using Abs to T cell surface markersor peptides specific for tumor antigens. Most studies conclude that theterminal effector cells responsible for T cell-specific tumor killingare CD8+ T cells. Therefore, prior strategies specifically augmented theCD8 arm of the immune system using MHC class I-specific tumor peptides.However, in certain animal tumor models that CD4+ T cells areresponsible for recognition and elimination of tumors. Augmentation oftumor-specific responses to both CD4 and CD8 T cells is the mostbeneficial tumor vaccine strategy.

[0753] Increasing the long-term survival of memory T cells afterimmunization is desirable for a successful vaccine. The generation oflarge numbers of memory T cells in vivo is difficult becauseAg-stimulated T cells are susceptible to activation-induced cell death.CD4 T cell arm of immunity is stimulated by the costimulatory receptorOX-40 (OX-40R) which is preferentially expressed by CD4+ T cells. OX40engagement results in a 60-fold increase in the number of Ag-specificCD4+ memory T cells that persisted 60 days postimmunization. Thus, OX40engagement early after immunization with soluble Ag enhances long-term Tcell and humoral immunity in a manner distinct from that provided byblocking CTLA-4.

[0754] The presentation of a tumor Ag to T cells by MHC class I or IImolecules is insufficient to prime an immune response in vivo. At leasttwo signals are necessary to activate a CD8 or CD4 T cell response. Thefirst signal is delivered through the T cell Ag receptor by Ag (peptide)bound to MHC class I or II. If only the first signal occurs, the T cellbecomes tolerant or undergoes apoptosis. A second signal involving theligation of a costimulatory molecule is required for optimal T cellactivation. The best-characterized second signal is delivered to the Tcell CD28 receptor by its ligand B7.1 or B7.2 (now known as CD80 andCD86, respectively). Both B7.1 and B7.2 bind to two determinants on Tcells, CD28 and CTLA-4; the former is constitutively expressed on 95% ofCD4+ and 50% of CD8+ T cells, and the latter is expressed only onactivated T cells. The interaction of CD80 or CD86 with CD28 results inincreased production of IL-2, which is necessary for the development ofa beneficial T cell response. In contrast, the interaction of B7 withCTLA-4 delivers a negative signal to the T cell and could negate theactivation signal of CD28.

[0755] The OX-40R is expressed on activated CD4+ T cells and whenengaged causes a potent costimulatory signal to effector T cells thatenhances long-term survival. Several other membrane-boundreceptor-ligand pairs serve as costimulators for T-cell activation. Inparticular, members of the tumor necrosis factor receptor superfamilyshare the ability to enhance or costimulate the process of T-cellactivation. This family consists of the CD30, CD40, CD27, Fas (CD95),DR3, 4-1BB, and OX-40. The 4-1BB receptor binds to a high-affinityligand (4-1BBL) expressed on several APCs such as dendritic cells,macrophages, and activated B cells. Expression of 4-1BB is somewhatrestricted to primed CD4, CD8 T cells, and natural killer cells. Ofparticular significance is that administration of 4-1BB mAb as a singleagent eradicates well-established tumors in mice. Although both CD4 andCD8 participated in the antitumor immune responses, the stimulation of aCD8CTL response was particularly striking. The CTL activity generatedfrom 4-1BB mAb-treated mice was increased up to approximately 65 timescompared with that of spleen cells from control animals. Thus, ligationof costimulation receptors in vivo may augment natural immunity to thegrowing tumors sufficient to induce their regression.

[0756] The OX-40 receptor (OX-40R) is a transmembrane protein found onthe surface of activated CD4(+) T cells. and not on normal restingperipheral blood lymphocytes. The OX-40 ligand (OX-40L) is a type IImembrane protein of ˜34,000 m.w., which is expressed on activated Bcells, activated endothelial cells, dendritic cells, and activatedmacrophages. It is not expressed on normal resting cells. The OX-40Ldelivers a potent costimulatory signal to OX-40R+T cells and anOX-40R/OX-40L interaction appears to be directly involved in an adhesionevent between endothelial cells and T cells. When engaged by an agonistsuch as anti-OX-40 antibody or the OX-40 ligand (OX-40L) during antigenpresentation to T cell lines, the OX-40R generates a costimulatorysignal that is as potent as CD28 costimulation. Engagement of OX-40Renhances effector and memory-effector T cell function by up-regulatingIL-2 production and increasing the life-span of effector T cells. Thesignal generated by the OX-40R inhibits activation-induced T cell death(AICD) and thereby increases the number of cells differentiating fromthe effector to memory T cell stage.

[0757] Previously, we showed that tumor cells transfected withsuperantigen SEB delivered with tumor cells transfected with thecostimulant B7 and MHC class II are effective in reducing the number oflung metastases in a metastatic breast cancer model, Example 70. Thepresent invention contemplates the transfection of tumor cells withOX-40 ligand gene (Godfrey W R et al., J. Exp. Med. 180: 757-762 (1994)or the high-affinity ligand (4-1BBL) or an Fv fragment that expressesanti-OX40R or anti-4-1BBL together with nucleic acids encoding asuperantigen. The gene for OX-40 ligand has been cloned and istransfected into tumor cells as described in Examples 1 and 70. Thesuperantigen gene is similarly transfected into the same tumor cells oralone into a equal number or the same tumor cells as given on pages 3-5and Example. The tumor cells cotransfected with SAg and OX-40 ligand orthe two populations of tumor cells transfected with superantigen andOX-40 ligand are injected into mice with known metastatic tumor as givenin Example 70. The results show that the mice treated with the tumorcells transfected with superantigen and OX-40 ligand had statisticallysignificant reduction in the number of metastases compared to mocktransfected tumor cells or either superantigen or OX-40 transfectedalone. The method of transfecting tumor cells with the costimulant genesand tumor models used are given in Examples 1 and 70. Similar resultsare obtained by cotransfecting high affinity 4-1BB1 ligand into tumorcells in place of OX-40 ligand. These transfectants used in the sametumor system also demonstrate a statistically significant reduction inthe number of lung metastases in the metastatic breast cancer model asdescribed in Example 70.

[0758] Superantigens Conjugated to OX40L or 4-1BBL and Tumor SpecificAntibody, Fab or scFv Fragments

[0759] The present invention contemplates the use of the superantigensand SETs in any form including but not limited to Superantigens and SETsof any kind including but not limited to staphylococcal enterotoxins A,B, C1, C3, C3, D, E, F, G H, I, J, K, L, M, streptococcal pyrogenicexotoxins, Yersinia pseudotuberculosis superantigen, M. arthitidissuperantigen, C. perfringens exotoxin A fused to a potent costimulantligand (extracellular domain preferred) such as OX-40 ligand or 4-11Bligand. A preferred superantigen in this conjugate is one to whichhumans do not make or make only marginal amounts neutralizing antibodyfused recombinantly or biochemically to a high affinity tumor specificantibody, Fab or single chain fv. Superantigens such as Yersiniapseudotuberculosis or clostridia perfringens toxin A to which humans donot have preexisting antibodies are preferred. One such superantigen,Yersinia pseudotuberculosis superantigen has, in addition, a natural RGDdomain with tumor localizing properties. Hence, Yersiniapseudotuberculosis is one of the preferred-superantigens for use in theSE-OX-40 ligand/4-11B ligand conjugate.

[0760] SEC1, is also preferred for use in the SE-OX-40 ligand or 4-11Bligand conjugate. The latter superantigen has the highest affinity forTCR Vβ regions of all superantigens. This TCR binding may bestrengthened in SEC mutants by amino acid substitutions in the Vβbinding areas of the molecule that will increase its TCR binding.Similarly, mutations in SEC MHC class II binding sequences may totallyeliminate SEC binding to MHC without interfering with its TCR binding,and activating function.

[0761] Another preferred superantigen for the SE-OX-40 ligand, 4-11Bligand conjugate is one with the deleted epitopes recognized byendogenous (preexisting) specific antibodies. These epitopes may bereplaced by amino acid substitutions producing sequences to which hostantibodies do not react. For example, a dominant epitope on SEBrecognized by anti-SEB antibodies is the sequence 225-234 (Nishi et al.,J. Immunol. 158: 247-254 (1997) and an epitope on SEA recognized byanti-SEA antibodies is the sequence 121-149 (Hobieka et al., Biochem.Biophys. Res. Comm. 223: 565-571 (1996).

[0762] Preferentially, the tumor targeting structure in superantigenconjugate (e.g., tumor specific antibody, Fab or single chain fvfragments or tumor receptor ligand) has a greater affinity for the tumorthan the SAg in the conjugate has for the MHC class II molecule thusfavoring binding of the conjugate to the tumor over binding to all cellscontaining MHC class II receptors. Ideally the molecule has thefollowing hierarchy of binding affinities: Tumor>TCR>MHC class II.

[0763] To further enhance the affinity of the tumor specific antibody inthe conjugate for tumor cells in vivo, the tumor specific antibodies areused which are specific for more than one antigenic structures on thetumor, tumor stroma or tumor vasculature or any combination thereof. Thetumor specific antibody or Fab′2, Fab or single chain Fv fragments aremono or divalent like IgG, polyvalent for maximal affinity like IgM orchimeric with multiple tumor (tumor stroma or tumor vasculature)specificities. Thus, when the SE or SPE-mAb conjugate is administered invivo, it will preferentially bind to tumor cells rather than toendogenous SE antibodies or MHC class II receptors.

[0764] To reduce affinity of the SE-mAb conjugate for endogenous MHCclass II binding sites, the high affinity Zn⁺⁺ dependent MHC class IIbinding sites in SEA, SEC2, SEC3, SED, SPEA, SPEC, SPEG, SPEH, SMEZ,SMEZ2, mycoplasma arthritides are deleted or replaced by inertsequence(s) or amino acid(s). These structural alterations in SE or SPEAreduce the affinity for MHC class II receptors from a K_(d) of 10⁻⁷ or10⁻⁸ to 10⁻⁵. SEB, SEC and SSA and other SEs or SPEs do not have a highaffinity Zn++ dependent MHC class II binding site but have multiple lowaffinity MHC class II binding sites (K_(d) 10⁻⁵-10⁻⁷). In these cases,alteration of the MHC class II binding sites is not always necessary tofurther reduce affinity for MHC class II receptors; at the very leastmutation of one or two of the low affinity MHC class II binding siteswill suffice in most instances.

[0765] Most importantly, tumor specific antibodies, Fab, F(ab)′2 orsingle chain Fab or Fv fragments in the SAg-mAb conjugate have a higheraffinity for tumor antigens (K_(d) 10⁻⁵-10⁻¹⁴ or lower) than for thesuperantigen has for MHC class II binding sites (K_(d) 10⁻⁵ to 10⁻⁷) andits dominant epitope has for superantigen specific antibodies (K_(d)10⁻⁷ to 10⁻¹¹). In this way, the conjugate will bind preferentially tothe tumor target in vivo rather than preexisting antibodies or MHC classII receptors.

[0766] SAg-OX-40 ligand (OX-40L) or 4-1BB ligand (4-1BBL) fused to atumor specific targeting structure is carried out using recombinantSAgs, the extracellular domains of OX-40L or 4-1BBL and the targetingstructure (high affinity tumor specific fv fragments are preferred). Thenucleic acids encoding the extracellular region of OX-40L (Godfrey etal., J. Exp. Med 180:757-762 (1994)) or 4-1BBL (Goodwin et al. Eur. J.Immunol. 23: 2631-2641 (1993); Melero I. et al., Eur. J. Immunol. 28:1116-1121 (1998)) are fused in frame with nucleic acids encoding a SAgof any type (Example 5). SEA, SEB, SEC and Y. pseudotuberculosis arepreferred. The SE may be modified to reduce antigenicity by modifying adominant epitope (e.g., SEB 227-232) and to reduce toxicity by alteringits MHC class II binding affinity (e.g., SEA D227A-high affinity Zn++dependent binding site). The tumor targeting structure may include butis not limited to a receptor ligand or antibody. The antibody can takethe form of F(ab)′₂, Fab, Fd or single chain fv fragments. Preferably,the affinity of the tumor targeting structure is of higher affinity forits tumor target than the affinity of the modified SAg for MHC class IIreceptors. High affinity single chain fv constructs that are specificfor the OX-40 receptor and 4-1BB receptor may be used in place of theOX40L and 4-1BBL in the SAg-tumor targeting construct.

[0767] The SE-OX-40L (or 4-1BB)-tumor specific Fv conjugate or SE-mAbFab-tumor-specific Fv conjugates described above are administeredparenterally, intratumorally, intrathecally, intraperitoneally,intrapleurally by infusion or injection in conventional or sustainedrelease vehicles as given in Section 66 in dosages of 0.01 ng/kg to 100μg/kg using protocols given in Examples 5, 7, 14, 15, 16, 18-23, 38.

[0768] Superantigens Conjugated to Anti-apoptotic Cytokines and TumorSpecific Antibody, Fab or Single Chain Fv Fragments

[0769] It is well established that superantigen stimulation of T cellscan result in activation driven cell death in vivo and in vitro. Severalcytokines and LPS are known to interdict this process. Cytokines IL-2,IL-4, IL-7 and IL-15 are known to prevent superantigen stimulated Tcells from undergoing apoptosis in vivo and in vitro (Vella et al.,Proc. Natl. Acad. Sci. 95: 3810-3815 (1998)). Accordingly,superantigen-mAb conjugate as described above is further fused to an Tcell anti-apoptotic cytokine. The extracellular domain of at least onecytokine from a group consisting of IL-2, IL-4, IL-7 and IL-15 is fusedrecombinantly to the SAg by methods given in Example 5. Other anti-Tcell apoptosis agents such as LPS preparations of low virulence or alipid A component (modified to induce less toxicity) are also effectiveantiapoptotic agents when conjugated biochemically to thesuperantigen-mAb (or F(ab)′₂, Fab, Fd or single chain fv fragments)conjugate or if administered concomitantly with the SAg.

[0770] The SE-antiapoptotic conjugates described above are administeredparenterally, intratumorally, intrathecally, intraperitoneally,intrapleurally by infusion or injection in conventional or sustainedrelease vehicles as given in Section 66 in dosages of 0.01 ng/kg to 100μg/kg using protocols given in Examples 5, 7, 14, 15, 16, 18-23, 38.

68. Superantigens for Treatment of Malignant Masses, Malignant Ascitesand Malignant Pleural Effusions

[0771] The present invention contemplates the use of the superantigensand SETs in any form including but not limited to Superantigens and SETsof any kind including but not limited to staphylococcal enterotoxins A,B, C, D, E, F, G H, I, J, K, L, M, streptococcal pyrogenic exotoxins, Y.pseudotuberculosis superantigen, M. arthitidis, C. perfringens exotoxinfor direct administration into cavities or spaces, e.g., peritoneum,thecal space, pericardial and pleural space containing tumor.

[0772] The present invention contemplates the direct injection of anysuperantigen although SEA, SEB and SEC are preferred into fluid spacescontaining tumor cells or adjacent to membranes such as pleural,peritoneal, pericardial and thecal spaces containing tumor. These sitesdisplay malignant ascites, pleural and pericardial effusions ormeningeal carcinomatosis. The superantigen is preferably administeredafter partial or complete drainage of the fluid (e.g., ascites, pleuralor pericardial effusion) but it may also be administered directly intothe undrained space containing the effusion, ascites and/orcarcinomatosis. In general, the SE dose is varied from 1 picogram to 10μg and given every 3 to 10 days. It is continued until there is noreaccumulation of the ascites or effusion. Therapeutic responses areconsidered to be no further accumulation of four weeks after the lastintrapleural administration.

[0773] In the US and Western Europe, there are 300,000 new cases ofmalignant pleural effusion annually. It is caused by lung cancer (35%),breast cancer (25%), lymphoma (10%), unknown primary malignancy (30%).It is the presenting manifestation in 10-50% of all cancers. Fiftypercent of cancer patients develop pleural effusion at some point intheir disease and up to 75% of MPEs are symptomatic on presentation. Itsappearance signifies Stage IIIb or IV disease. The prognosis is 3-4months.

[0774] Malignant ascites is associated with ovarian tumors (30-50%).Endometrial, breast, colonic, gastric and pancreatic carcinomas make upmore than 80% or the tumors associated with intra-abdominal seeding andascites formation. Ascites may be the presenting manifestation in 4-69%of cases.

[0775] The major therapies for malignant pleural effusion include talcpoudrage, talc slurry, doxycycline and bleomycin instillation. Thesetherapies require 3-12 days of hospitalization with EKG and oximetrymonitoring. A chest tube is inserted and the medication is infused andallowed to distribute over the pleural membranes. The chest tube is thenconnected to closed negative pressure water seal drainage until pleuralfluid volume is <10° C.c/24 hours. Respiratory therapy is usually givenat least once daily.

[0776] Talc poudrage requires the use of operating room for thoracostomyand talc insufflation, general anesthesia and recovery room observation.Talc suffers from respiratory complications in up to 33% of patients andacute respiratory distress and hypoxemia in 10% of patients. Bleomycinand Doxycycline have response rates of 50% and 70% respectively and bothrequire continuous chest tube drainage until the output is <100 cc/24hours.

[0777] Intrapleurally administered chemotherapeutic agents Cisplatin,Cytarabine, fluorouracil, Etoposide, Mitomycin C, radiation and biologicagents IL-2, IFN-α and β, Corynebacterium parvum have been ineffectiveagainst MPEs. Thoracentesis or chest tube drainage alone results inrecurrence rates of 98% and 85% respectively within 30 days.Intraperitoneal cisplatin and etoposide has produced a complete responserate of 30% of malignant ascites. However the only randomized study hasfailed to show any benefit for intraperitoneal therapy over conventionalintravenous chemotherapy in the initial management of stage II C to IVovarian cancer. No definitive success of various biologic agents, e.g.,IFN-α, β, and γ, TNF or IL-2. Systemic chemotherapy in ascitesassociated with ovarian carcinoma can achieve a 5 year survival of 50%in stage I and II.

[0778] In contrast to Bleomycin and Doxycycline, superantigeninstillation into the pleural space has a response rate of near 100% anddoes not require continuous chest tube drainage until the 24 hour outputis <10° C.c. The superantigen is administered immediately after removalof pleural fluid via thoracentesis. On an average, this procedure isrepeated once weekly for three treatments after which there is generallyno further fluid accumulation. It is carried out entirely in anoutpatient setting and requires no hospitalization at a cost severalhundred percent below that of existing therapy. Major costs of the othertherapies originating from hospitalization, chest tube insertion,operating and recovery room expense, respiratory therapy and in-hospitalchest tube drainage, are eliminated.

[0779] Patients with malignant pleural effusions confirmed by biopsy orpleural fluid cytology are entered. They have not been undergoing anyother anticancer treatment for at least one month and have a clinicalKarnofsky status of at least 60-70%. SEA in doses of 10-30 ng isadministered intrapleurally once or twice weekly immediately afterdrainage of the effusion via conventional thoracentesis. This procedureis performed once or twice weekly in an outpatient or office setting.Treatment is continued once weekly until effusion does not recur.

[0780] An objective response is recognized as no reaccumulation ofpleural fluid 30 days after treatment (DeCamp M M et al., Chest 112:291S-295S (1997); Fenton K N et al., Am J. Surg. 170: 69-74 (1995)).

[0781] There are 90 evaluable patients with malignant pleural effusionstreated with intrapleural SEA. All patients have stage IIIb or stage IVlung cancer. There are 50 evaluable patients with malignant ascites.

[0782] Eighty five patients with pleural effusions exhibit objectiveclinical responses for a response rate of 94.5%. Effusion reaccumulation(at weekly intervals) progressively diminished after each SEA treatment.An example of progressive reduction of effusion reaccumulation aftereach treatment is shown below. Patients required an average of threetreatments before there was no significant reaccumulation. However,several patients require only one treatment to eliminate fluidreaccumulation. Forty five patients with malignant ascites showobjective responses for a response rate of 90%.

[0783] Toxicity in both malignant pleural effusion and ascites consistsof mild short-lived fever, fatigue and anorexia not requiring treatment.CBC, renal and liver functions tests did not change significantly aftertreatments.

[0784] The superantigen has better therapeutic efficacy for malignantpleural effusions and ascites without the discomfort and complicationsassociated with an indwelling draining chest tube. In the case ofpleural effusion, It is also 90% more cost-effective compared toexisting therapy since it is carried out in an outpatient facility anddoes not involve the major costs associated with hospitalization, i.e.,chest tube insertion, operating and recovery room, indwelling chest tubedrainage and respiratory therapy.

69. Intratumorally Administered Superantigens

[0785] The present invention contemplates the use of the superantigensand SETs in any form including but not limited to Superantigens and SETsof any kind including but not limited to staphylococcal enterotoxins A,B, C, D, E, F, G H, I, J, K, L, M, streptococcal pyrogenic exotoxins, Y.pseudotuberculosis superantigen, M. arthitidis superantigen, C.perfringens exotoxin for direct intratumoral treatment of tumor masses.Tumor masses appearing in any organ, palpated or visualized on x-ray, CTscan or MRI ultrasound are injected directly with any of thesuperantigens.

[0786] The superantigen is administered by injection or infusiondirectly into the tumor mass and is carried out with fluoroscopic, CTguidance or ultrasound guidance. For intratumoral administration, SE isdissolved in a conventional fluid vehicle such as saline or it may beincorporated into a controlled release formulation (mixture orsuspension) preferably biodegradable. All of the biocompatible andbiodegradable and controlled release formulations given in section 66are useful. These formulations also include but are not limited to,ethylene-vinyl acetate (EVAc: Elvax 40W, Dupont), bioerodiblepolyanhydrides, polyimino carbonate, sodium alginate microspheres andhydrogels. Dosages used range from 1 ng to 10 mg. The poly-(D-, L- orDL-lactic acid/polyglycolide) copolymers are preferred.

[0787] Use of Intratumoral Superantigen with Chemotherapy

[0788] The SE are used for intratumoral injection. This is done withfluoroscopic, CT or ultrasound guidance. For intratumoraladministration, SE is dissolved in a conventional fluid vehicle such asor it may be incorporated into a controlled release formulation (mixtureor suspension) preferably biodegradable. All of the biodegradablecontrolled release formulations given in section 64 are useful althoughthe poly d-1 glycolides are preferred because of biocompatibility andbiodegradability.

[0789] A variety of chemotherapeutic agents may be used in the combinedtreatment methods disclosed herein. Chemotherapeutic agents contemplatedas exemplary include, e.g., cisplatin (CDDP), adriamycin, actinomycin,mitomycin, carminomycin, daunomycin, doxorubicin, tamoxifen, taxol,taxotere, vincristine, vinblastine, vinorelbine, etoposide (VP-16),5-fluorouracil (5FU), cytosine arabinoside, cyclophosphamide, thiotepa,methotrexate, campothecin, actinomycin-D, mitomycin C, aminopterin,combretastatin(s) and derivatives and prodrugs thereof. Anti-cancerchemotherapeutic drugs useful in this invention include but are notlimited to antimetabolites, anthracycline, vinca alkaloid, anti-tubulindrug, antibiotic, alkylating agent.

[0790] The chemotherapeutic agent(s) selected for use preferably showsthe highest response rate against tumor to be treated. For example, innon-small cell lung cancer, the cisplatin-based trials showed a benefitof chemotherapy with a hazard ratio of 0.73 (p<0.0001), equivalent to anabsolute improvement in survival of 10% (5-15%) at 1 year, or anincrease in median survival of 1½ months (1-2½ months).Completedprospective randomized trials including quality-of-life analyses showthat cisplatin-based therapeutic regimens also improve quality of lifein these patients. Other agents in phase III trials in patients withadvanced NSCLC include the taxanes (paclitaxel and docetaxel), vincaalkaloid (vinorelbine), antimetabolite (gemcitabine), and campothecin(irinotecan). These agents have shown promise in both phase I and IItrials, both as single agents and in combination with a platinum agent.

[0791] Example 73 provides the chemotherapeutic agents preferred for usein various malignancies. These agents would be used as single agents orcombined with other agents in full doses or in doses 10-95% below therecommended dose. They may be delivered intratumorally alone or togetherwith SE in doses 5-50% (e.g., 5-10 mg weekly for 2-7 weeks) below thesystemic dose of the drug per cycle. Cisplatin is preferentiallydelivered intratumorally in a small volume (2-5 ml) of saline whichproduces a highly viscous solution that has the benefit of beingretained for sustain periods in the tumor. The SE can be administeredtogether with the cisplatin in the same or separate syringe or in twoseparate syringes. Preferred tumors to treat are solitary nodules inorgans located close to the skin surface including but not limited tohepatocellular carcinoma, lung tumors, brain tumors, head and necktumors, unresectable breast tumors. The optimal chemotherapeutic agentsand combined regimens for all the major human tumors are set forth inBethesda Handbook of Clinical Oncology, Abraham J et al., LippincottWilliam & Wilkins, Philadelphia, Pa. (2001); Manual of ClinicalOncology, Fourth Edition, Casciato, D A, Lowitz, B B, Lippincott William& Wilkins, Philadelphia, Pa. (2000) which are herein incorporated inentirety by reference.

[0792] As will be understood by those of ordinary skill in the art, theappropriate doses of chemotherapeutic agents will be generally aroundthose already employed in clinical therapies wherein thechemotherapeutics are administered alone or in combination with otherchemotherapeutics. By way of example only, agents such as cisplatin, andother DNA alkylating may be used. Cisplatin has been widely used totreat cancer, with efficacious doses used in clinical applications of 20mg/m² for 5 days every three weeks for a total of three courses.Cisplatin is not absorbed orally and must therefore be delivered viainjection intravenously, subcutaneously, intratumorally orintraperitoneally.

[0793] Further useful agents include compounds that interfere with DNAreplication, mitosis and chromosomal segregation. Such chemotherapeuticcompounds include adriamycin, also-known as doxorubicin, etoposide,verapamil, podophyllotoxin, and the like. Widely used in a clinicalsetting for the treatment of neoplasms, these compounds are administeredthrough bolus injections intravenously at doses ranging from 25-75 mg/m²at 21 day intervals for adriamycin, to 35-50 mg/m² for etoposideintravenously or double the intravenous dose orally.

[0794] Agents that disrupt the synthesis and fidelity of polynucleotideprecursors may also be used. Particularly useful are agents that haveundergone extensive testing and are readily available. As such, agentssuch as 5-fluorouracil (5-FU) are preferentially used by neoplastictissue, making this agent particularly useful for targeting toneoplastic cells. Although quite toxic, 5-FU, is applicable in a widerange of carriers, including topical, however intravenous administrationwith doses ranging from 3 to 15 mg/kg/day being commonly used

[0795] In non-small cell lung cancer, the cisplatin-based trials showedan absolute improvement in survival of 10% (5-15%) at 1 year, or anincrease in median survival of 1½ months (1-2½ months). Cisplatin-basedtherapeutic regimens also improve quality of life in these patients.Other agents in phase III trials in patients with advanced NSCLC includethe taxanes (paclitaxel and docetaxel), vinca alkaloid (vinorelbine),antimetabolite (gemcitabine), and camptothecin (irinotecan). Theseagents have shown promise in both phase I and II trials, both as singleagents and in combination with a platinum agent. One-year survival ratesof up to 40% have been commonly reported.

[0796] Suggested single and combination chemotherapy regimens fornon-small cell lung cancer, breast cancer and hepatocellular cancer andchemotherapeutic agents commonly used for various malignancies are givenin Example 73.

[0797] Other agents and therapies that are operable after intratumoralsuperantigen include, radiotherapeutic agents, antitumor antibodies withattached anti-tumor agents such as plant fungus or bacteria-derivedtoxin or coagulant, ricin A chain, deglycosylated ricin A chain, aribosome inactivating protein, a-sarcin, gelonin, aspergillin,restricticin, a ribonuclease, a epipodophillotoxin, diphtheria toxin, orPseudomonas exotoxin. Additional cytotoxic, cytostatic or anti-cellularagents capable of killing or suppressing the growth or cell division ofthe tumor being treated include anti-angiogenic agents,apoptosis-inducing agents, coagulants, prodrug or tumor targeted form,tyrosine kinase inhibitors (Siemeister et al., 1998), antisensestrategies, RNA aptamers and ribozymes against VEGF or VEGF receptors(Saleh et al., 1996; Cheng et al, 1996; Ke et al., 1998; Parry et al.,1999; each incorporated herein by reference).

[0798] Numerous tyrosine kinase inhibitors useful. These include, forexample, the 4-aminopyrrolo[2,3-d]pyrimidines of U.S. Pat. No.5,639,757, specifically incorporated herein by reference, which may alsobe used in combination with the present invention. Further examples oforganic molecules capable of modulating tyrosine kinase signaltransduction via the VEGFR2 receptor are the quinazoline compounds andcompositions of U.S. Pat. No. 5,792,771, which is specificallyincorporated herein by reference for the purpose of describing furthercombinations for use with the present invention in the treatment ofangiogenic diseases.

[0799] Other agents such as steroids including the angiostatic4,9(11)-steroids and C21-oxygenated steroids, as described in U.S. Pat.No. 5,972,922, specifically incorporated herein by reference, may beemployed in combined therapy. U.S. Pat. Nos. 5,712,291 and 5,593,990,each specifically incorporated herein by reference, describe thalidomideand related compounds, precursors, analogs, metabolites and hydrolysisproducts, which may also be used in combination with the presentinvention to inhibit angiogenesis. The compounds in U.S. Pat. Nos.5,712,291 and 5,593,990 can be administered orally.

[0800] Certain anti-angiogenic therapies have already been shown tocause tumor regressions, including the bacterial polysaccharide CM101and the antibody LM609. CM101 is a bacterial polysaccharide that hasbeen well characterized in its ability to induce neovascularinflammation in tumors. CM101 binds to and cross-links receptorsexpressed on dedifferentiated endothelium that stimulates the activationof the complement system. It also initiates a cytokine-driveninflammatory response that selectively targets the tumor. It is auniquely antipathoangiogenic agent that downregulates the expressionVEGF and its receptors. CM101 is currently in clinical trials as ananti-cancer drug, and can be used in combination herewith.

[0801] Thrombospondin (TSP-1) and platelet factor 4 (PF4) may also beused in combination with the present invention. These are bothangiogenesis inhibitors that associate with heparin and are found inplatelet .alpha.-granules. TSP-1 is a large 450 kDa multi-domainglycoprotein that is constituent of the extracellular matrix. TSP-1binds to many of the proteoglycan molecules found in the extracellularmatrix including, HSPs, fibronectin, laminin, and different types ofcollagen. TSP-1 inhibits endothelial cell migration and proliferation invitro and angiogenesis in vivo. TSP-1 can also suppress the malignantphenotype and tumorigenesis of transformed endothelial cells. The tumorsuppressor gene p53 has been shown to directly regulate the expressionof TSP-1 such that, loss of p53 activity causes a dramatic reduction inTSP-1 production and a concomitant increase in tumor initiatedangiogenesis.

[0802] PF4 is a 70aa protein that is member of the CXC ELR-family ofchemokines that is able to potently inhibit endothelial cellproliferation in vitro and angiogenesis in vivo. PF4 administeredintratumorally or delivered by an adenoviral vector is able to cause aninhibition of tumor growth.

[0803] Interferons and metalloproteinase inhibitors are two otherclasses of naturally occurring angiogenic inhibitors that can becombined with the present invention. The anti-endothelial activity ofthe interferons has been known since the early 1980s, however, themechanism of inhibition is still unclear. It is known that they caninhibit endothelial cell migration and that they do have someanti-angiogenic activity in vivo that is possibly mediated by an abilityto inhibit the production of angiogenic promoters by tumor cells.Vascular tumors in particular are sensitive to interferon, for example,proliferating hemangiomas can be successfully treated with IFNα.

[0804] Tissue inhibitors of metalloproteinases (TIMPs) are a family ofnaturally occurring inhibitors of matrix metalloproteases (MMPs) thatcan also inhibit angiogenesis and can be used in combined treatmentprotocols. MMPs play a key role in the angiogenic process as theydegrade the matrix through which endothelial cells and fibroblastsmigrate when extending or remodeling the vascular network. In fact, onemember of the MMPs, MMP-2, has been shown to associate with activatedendothelium through the integrin .alpha.v.beta.3 presumably for thispurpose. If this interaction is disrupted by a fragment of MMP-2, thenangiogenesis is downregulated and tumor growth is inhibited.

[0805] SEA-Chemotherapy Outcomes

[0806] Patients with malignant masses confirmed by biopsy or cytologyare entered. They have not been undergoing any other anticancertreatment for at least one month and have a clinical Karnofsky status ofat least 60-70%. SEA is used as the prototypical SAg but other SAgs andhomologues as defined in Section 62 can be used as well in comparabledoses. SEA is administered in doses of 0.01 ng-100 ug/kg ngintratumorally once every 2-7 days. The tumors are accessiblepercutaneously. Others may be visualized and treated via bronchoscopy orstereotactically in the case of brain tumors. The low dose chemotherapyis started preferably within 36 hours after the last treatment accordingto protocols in Example 73. Chemotherapy may be started at the same timeas immunotherapy, however, it is preferable to start chemotherapy within36 hours after the second to fourth dose of SEA. Chemotherapy is givenpreferably in subtherapeutic doses (10-95% below the FDA approved dosageper cycle) but full therapeutic doses are also useful. Responses areclassified according to the WHO and RECIST criteria in Abraham et al.,supra. Patients receive two and eight weeks of therapy.

[0807] There are 100 evaluable patients with malignant masses treatedwith intratumoral SEA. All patients have stage IIIb or stage IV lungcancer. Eighty five patients exhibit objective clinical responses for aresponse rate of 85%. Tumors generally start to diminish and objectiveremissions are evident after four weeks of combined SEA-chemotherapy.Responses endure for an average of 24 months.

[0808] Toxicity consists of mild short-lived fever, fatigue and anorexianot requiring treatment. CBC, renal and liver functions tests do notchange significantly after treatments. The SAg—chemotherapy has betterresponse rate and duration of remission than either agent alone withminimal toxicity.

69. Use of Superantigens as a Vaccine or against Established InfectiousDisease

[0809] The present invention contemplates the use of superantigens inany form to combat or boost host immunity to infectious diseases. Thesuperantigen is used as a vaccine and is delivered together with orwithin 12 days after the administration of the infectious agent or acomponent (antigenic structure) of the infectious agent, a homologue orfragment of the infectious agent or a surrogate or the infectious agent.The infectious agent is a virus, bacterium, fungus. Parasite orprotozoan. The superantigen is also used to treat established infectiousdiseases. For example, a superantigen is administered directly into atuberculosis pleural effusion or ascites or directly into a tuberculousgranuloma or abscess. For use as a vaccine, the superantigen isadministered parenterally by infusion or injection. It is alsoadministered intrathecally, intrapleurally or intrapertioncally. It isdissolved in conventional diluents known in the art or in sustainedrelease vehicles as given in section 66. For use in an establishedinfection such as an empyema, pleural effusion or ascites of bacterial,fungal or tuberculous origin, superantigen is preferentially givenintrapleurally or intraperitoneally respectively. Doses of thesuperantigen given in soluble form are 1 ng to 50 μg and in sustainedrelease preparations, 1 ng to 50 mgs. The superantigen in soluble formis administered once every 3-10 days for 2-7 treatments until there aresigns that the pleural effusion or ascites is not reaccumulating and agranuloma or abscess is regressing. When used in sustained releasepreparations against established infection, the superantigen isadministered once weekly for 3-7 treatments of once monthly for 3-7months.

[0810] In all of these situations, the costimulant ligands OX-40 and4-11B are administered together with the superantigen and up to 5 daysafterward by injection or infusion by intravenous, intramuscular,subcutaneous, intrapleural, intraperitoneal, intralesional orintrathecal routes in doses of 0.2 ng/kg to 20 m/kg for each treatment.

70. Amplification of the α-1,4-Digalacosyl-Ceramide Binding Unit of SAg

[0811] Abbreviations. GaOse₂ or galabiosylceramide,Gal(α1-4)Gal/β1-ceramide: GbOse₃, or globotriaosylceramideGal(α1-4)Gal(β1-4)Glcβ-1 ceramide; GbOse₄ or globotetraosyl ceramide.GalNac(β1-3)Gal(α1-4)Gal(β1-4)Glc ceramide.

[0812] The structure of the amino-terminal domain of SEs closelyresembles the oligosaccharide/oligonucleotide-binding fold (OB fold)characteristic of verotoxin-1, cholera toxin, pertussis toxin, AB5toxins from E. coli and the active domain of metalloprotease-2. Thesuperantigen SEB binds to the GaOse₂ or galabiosylceramide receptor oncultured human proximal tubular epithelial cells (Chatterjee et al.,Glycobiology 5:327-333 (1995)). GaOse₂ and Globotriaosylceramide(GbOse₃) are recognized by human verotoxins (verotoxin-1, verotoxin-2and verotoxin 2c) (Boyd et al. Eur. J Biochem. 223:873-8 (1994)). It isessential that the Gal(α1-4)Gal is terminal on the carbohydrate sequenceto permit verotoxin binding since digestion of the glycolipids withα-galactosidase resulted in loss of toxin binding. In TLC overlayexperiments, the GaOse₂ is the most effective verotoxin receptor.Verotoxin binds to astrocytoma cells, ovarian carcinoma and Burkitt'slymphoma cells (Farkas-IIimsley et at., Proc. Natl. Acad Sci. 92:6996-7000 (1995); Arab et al., J. Neuro-Onc. 40: 137-150 (1998))presumably via the GbOse₃ and induces apoptosis. Several SEs are knownto bind to several MHC class II negative tumors such as colonadenocarcinomas and squamous cell carcinoma cells and induce T cellmitogenesis or SDCC (Dohlsten et al., Eur. J. Immunol. 21:1229 (1991);Hermann et al., J. Immunol. 146:2504-2512 (1991); Lamphear et al. J.Immunol. 160:615 (1998)). In the present invention, SAgs bind to GaOse₂receptor moieties overexpressed on carcinoma cells and other malignantcells just as ovarian carcinoma cells express 1000 fold more GbOse₃ thannormal ovarian cells.

[0813] In the present invention, the OB fold of the verotoxin-1 (anyother verotoxin) is fused to the native SE molecule. SEB, SEC1, SED andSEA are preferred superantigens for this purpose. The addition of thismoiety confers the SE with enhanced recognition of the GaOse₂ structureson target tumor cells. The fusion of the verotoxin OB fold to the SE isaccomplished recombinantly fusing the verotoxin GaOse₂ coding region (inits OB fold) to SEB preferably at its C terminus (away from the OB foldin the amino terminal domain). In an additional embodiment, the OB foldof the superantigen is duplicated to provide additional ligand forbinding to tumor cells expressing the receptors for this ligand.

[0814] In an additional embodiment, the Shiga toxin A subunit is fusedto the superantigen. The A subunit of the verotoxin is capable ofbinding to verotoxin receptors on tumor cells where it is internalizedand cleaved to a product that interferes with post translational ADPribosylation.

[0815] The SE-verotoxin conjugate is used to treat tumors or enhanceantitumor immunity. For this purpose it is administered parenterally,intratumorally, intrathecally, intraperitoneally, intrapleurally byinfusion or injection in conventional or sustained release vehicles asgiven in Section 66 in dosages of 0.01 ng/kg to 100 μg/kg usingprotocols given in Examples 5, 7, 14, 15, 16, 18-23, 38.

71. Coaguligands: SEs Fused to Coagulation Factors

[0816] Superantigens may be conjugated to, or operatively associatedwith, polypeptides that are capable of directly or indirectlystimulating coagulation, thus forming a “coaguligand” (Barinaga M etal., Science 275:482-4 (1997); Huang X et al., Science 275:547-50(1997); Ran S et al., Cancer Res Oct. 15 1998;58(20):4646-53; GottsteinC et al., Biotechniques 30:190-4 (2001)).

[0817] In coaguligands, the antibody may be directly linked to a director indirect coagulation factor, or may be linked to a second bindingregion that binds and then releases a direct or indirect coagulationfactor. The ‘second binding region’ approach generally uses acoagulant-binding antibody as a second binding region, thus resulting ina bispecific antibody construct. The preparation and use of bispecificantibodies in general is well known in the art, and is further disclosedherein.

[0818] Coaguligands are prepared by recombinant expression. The nucleicacid sequences encoding the SAg are linked, in-frame, to nucleic acidsequences encoding the chosen coagulant, to create an expression unit orvector. Recombinant expression results in translation of the new nucleicacid, to yield the desired protein product.

[0819] Where coagulation factors are used in connection with the presentinvention, any covalent linkage to the SAg should be made at a sitedistinct from the functional coagulating site. The compositions are thus“linked” in any operative manner that allows each region to perform itsintended function without significant impairment. Thus, the SAg binds toand stimulates T cells, and the coagulation factor promotes bloodclotting.

[0820] Preferred coagulation factors are Tissue Factor (“TF)compositions, such as truncated TF (tTF), dimeric, multimeric and mutantTF molecules. “Truncated TF” (tTF) refers to TF constructs that arerendered membrane-binding deficient by removal of sufficient amino acidsequences to effect this change in property. A “sufficient amount” inthis context is an amount of transmembrane amino acid sequenceoriginally sufficient to enter the TF molecule in the membrane, orotherwise mediate functional membrane binding of the TF protein. Theremoval of such a “sufficient amount of transmembrane spanning sequence”therefore creates a truncated TF protein or polypeptide deficient inphospholipid membrane binding capacity, such that the protein issubstantially a soluble protein that does not significantly bind tophospholipid membranes. Truncated TF thus substantially fails to convertFactor VII to Factor VIIa in a standard TF assay, and yet retainsso-called catalytic activity including activating Factor X in thepresence of Factor VIIa.

[0821] U.S. Pat. No. 5,504,067 is specifically incorporated herein byreference describes truncated TF proteins. Preferably, the TFs for useherein will generally lack the transmembrane and cytosolic regions(amino acids 220-263) of the protein. However, there is no need for thetruncated TF molecules to be limited to molecules of the exact length of219 amino acids.

[0822] Any of the truncated, mutated or other TF constructs maybeprepared in a dimeric form. TF dimers are prepared by employing thestandard techniques of molecular biology and recombinant expression, inwhich two coding regions are prepared in-frame and expressed from anexpression vector. Equally, various chemical conjugation technologiesmay be employed to prepare TF dimers. The individual TF monomers may bederivatized prior to conjugation.

[0823] The tTF constructs may be multimeric or polymeric. A “polymericconstruct” contains 3 or more Tissue Factor constructs. A “multimeric orpolymeric TF construct” is a construct that comprises a first TFmolecule or derivative operatively attached to at least a second and athird TF molecule or derivative. The multimers may comprise betweenabout 3 and about 20 such TF molecules. The constructs may be readilymade using either recombinant manipulation and expression or usingstandard synthetic chemistry.

[0824] TF mutants deficient in the ability to activate Factor VII areuseful. Such “Factor VII activation mutants” are generally definedherein as TF mutants that bind functional Factor VII/VIIa,proteolytically activate Factor X, but are substantially free from theability to proteolytically activate Factor VII. Accordingly, suchconstructs are TF mutants that lack Factor VII activation activity.

[0825] The ability of such Factor VII activation mutants to function inpromoting tumor-specific coagulation is based upon their specificdelivery to the tumor vasculature, and the presence of Factor VIIa atlow levels in plasma. Upon administration of such a Factor VIIactivation mutant conjugate, the mutant will be localized within thevasculature of a vascularized tumor. Prior to localization, the TFmutant would be generally unable to promote coagulation in any otherbody sites, on the basis of its inability to convert Factor VII toFactor VIIa. However, upon localization and accumulation within thetumor region, the mutant will then encounter sufficient Factor VIIa fromthe plasma in order to initiate the extrinsic coagulation pathway,leading to tumor-specific thrombosis. Exogenous Factor VIIa could alsobe administered to the patient.

[0826] Any one or more of a variety of Factor VII activation mutants maybe prepared and used in connection with the present invention. TheFactor VII activation region generally lies between about amino acid 157and about amino acid 167 of the TF molecule. Residues outside thisregion may also prove to be relevant to the Factor VII activatingactivity. Mutations are inserted into any one or more of the residuesgenerally located between about amino acid 106 and about amino acid 209of the TF sequence (WO 94/07515; WO 94/28017; each incorporated hereinby reference).

[0827] A variety of other coagulation factors may be used in connectionwith the present invention, as exemplified by the agents set forthbelow. Thrombin, Factor V/Va and derivatives, Factor VIII/VIIIa andderivatives, Factor IX/IXa and derivatives, Factor X/Xa and derivatives,Factor XI/XIa and derivatives, Factor XII/XIIa and derivatives, FactorXIII/XIIIa and derivatives, Factor X activator and Factor V activatormay be used in the present invention.

[0828] These conjugates are administered intrathecally by in dosages of0.01 ng/kg to 100 μg/kg.

EXAMPLE 1 Preparation of Plasmids for making DNA Templates for any Geneof Interest and the Process of Transfection

[0829] Mammalian oncogenes, and genes for oncogenic transcriptionfactors, angiogenic factors, growth factor receptors and amplicons aswell as bacterial and SAg plasmids and DNA are prepared as described inthe text references. When necessary, they are modified to forms suitablefor transfection into mammalian tumor cells or accessory cells usingmethods well described in the art. (Old R W et al., Principles of GeneManipulation, 5^(th) Ed., Blackwell 1994).

[0830] As a representative SAg, enterotoxin B plasmid DNA is prepared bythe method of Jones C L et al., J. Bacteriology 166 29-33 (1986) andRanelli et al., Proc. Natl. Acad. Sci. USA 82:5850-5854 (1985) using theCsCl-ethidium bromide density gradient centrifugation of cleared lysatesas described (Clewell, D B et al., Proc. Natl. Acad. Sci. USA62-1159-1166 (1969)). S. aureus chromosomal DNA was isolated asdescribed by Betley M et al., Proc. Natl. Acad. Sci. USA 81: 5179-5183(1984). E. coli HB101 was transformed with plasmid DNA by the CaCl2procedure of Morrison D A et al., Meth. Enzymol. 68:326-331 (1979).Restriction digests were analyzed by 1% agarose and 5% acrylamide gelelectrophoresis using Tris/Borate/EDTA buffer as described in Greene P Jet al., Methods Mol Biol. 7: 87-111 (1974). Additional methods forisolation and cloning of specific bacterial and mammalian plasmid DNAuseful in tumor or accessory cell transfection are cited in referencesgiven previously in the text or in Snyder L et al., Molecular Geneticsof Bacteria, ASM Press, Washington, D.C. (1997); Peters et al., supra;Franks et al., supra.

[0831] Suitable template DNA for production of mRNA encoding a desiredpolypeptide may be prepared using standard recombinant DNA methodologyas described in Ausubel F et al. Short Protocols in Molecular Biology3^(rd) Ed. John Wiley, New York, N.Y. (1995). There are numerousavailable cloning vectors and any cDNA containing an initiation codoncan be introduced into the selected plasmid and mRNA can be preparedfrom the resulting template DNA. The plasmid can be cut with anappropriate restriction enzyme to insert any desired cDNA coding for apolypeptide of interest. For example the readily available cloningvector pSP64T can be used after linearization and transcription with SP6RNA polymerase. Smaller sequence may be inserted into the Hind III/EcoTIfragment with T4 ligase. Resulting plasmids are screened for orientationand transformed into E. coli. These plasmids are adapted to receive anygene of interest at a unique BglII restriction site which is placedbetween the two Xenopus β-globin sequences.

[0832] Subcloning of SEB into pHβ-Apr-1-Neo Expression Vector

[0833] The Staphylococcal enterotoxin B (SEB) gene has been subclonedinto pHβ-Apr-1-neo expression vector. The final construct contained onlythe coding sequence of SEB: and conferred resistance to ampicillin andG-418.

[0834] Materials and Methods

[0835] PCR:

[0836] 1. The following two primers are designed and made at LifeTechnologies, Inc.: Primer SEB 1: total 24 bp 5′ to 3′GGCGTCGACATGTATAAGAGATTA SalI site Primer SEB2: total 24 bp 5′ to 3′GCCGGATCCTCACTTTTTCTTTGT BamHI site Both primers were dissolved infilter-sterilized ddH2O to a final concentration of 20 mM (stocksolution).

[0837] 2.The volume (in ml) of reagents for each PCR reaction is listedbelow: Exp. Exp. Exp. Exp. Exp. Reagent 1 2 3 4 5 ddH2O 76 72 67 49 5910 × PCR buffer 10 10 10 10 10 10 × dNTP(2 mM stock) 10 10 10 10 10Primer SEB1 (20 mM stock) 1 5 1 10 10 Primer SEB2 (20 mM stock) 1 1 1 1010 SEE Template (50 mg stock) 1 1 10 10 0 Pfu Turbo Enz 1 1 1 1 1 FinalVolume 100 100 100 100 100

[0838] 3. The following cycling parameters were applied:

[0839] 95° C. 1 minute 1 cycle initial denature

[0840] 95° C. 45 seconds denature

[0841] 52° C. 1 minute 20 cycles anneal

[0842] 72° C. 1 minute extension

[0843] 72° C. 1 minute 1 cycle final extension

[0844] 4° C. hold

[0845] 4. To verify that the PCR reactions yielded the correct sizefragment, 10 ml of the reaction mixture was electrophoresed on a 1%agarose gel in 1×TAE buffer.

[0846] Vector

[0847] 1. The pHβ-Apr-1-neo expression vector was spotted the vector ona filter paper. See FIG. 1

[0848] 2. To recover the DNA, the circle was cut out and added to 100 mlof H₂O to allow rehydration for 5 minutes. After a brief centrifugation,the supernatant was used to transform E. coli XL 1Blue (Stratagene), andselected by ampicillin (final concentration 100 mg/ml).

[0849] 3. To verify that the vector is correct, 4 ampR clones wererandomly selected and the clones were cultured in LB amp media. DNA wasisolated and digested with SalI, BamHI (single digest) and EcoRI/HindIII(double digest). The digested products were electrophoresed on a 1%agarose gel in 1×TAE buffer. The profile of the restriction digestconfirmed that the vector is correct.

[0850] Cloning and Verification

[0851] 1. The correct PCR fragments in experiments 2, 3, and 4 werepooled and gel-purified. A portion of the fragments was digested withrestriction enzymes SalI and BamHI, and was ligated into the digestedpHβ-Apr-1-neo expression vector. The ligation products were transformedinto E. coli XL1Blue (Stratagene). Insert containing clones wereselected by ampicillin.

[0852] 2. Ten ampicillin resistant clones were randomly selected,cultured in 5 ml of LB amp media, and their plasmid DNA was isolated.Insert containing clones (SEB construct) were verified by digesting theDNA with SalI and BamHI restriction endonucleases and electrophoresis at0.8% agarose gel. (See FIG. 2)

[0853] 3. One of the SEB constructs (clone #2) was verified bysequencing and aligned with the published SEB sequence in the “SEB andClone #2 Coding Sequence Table” below, which shows alignment of thepublished SEB coding sequence and the newly constructed SEB gene inpHβ-Apr1 neo vector (Clone #2). Clone #2 was sequenced with 4 primers:SEB1, 2, 3, and 4. SEB1 and 2 are the PCR primers that were used for theamplification of the SEB gene.

[0854] SEB 3 (TATGAAAGTTTTGTATGATGAT) and SEB 4 (AGTGACGAGTTAGGTAATCT)are internal primers. The final sequence was confirmed by the multipleoverlapping of sequences and aligned with the published SEB sequence. Itis a perfect match. The start codon (ATG) and the stop codon (TGA) areunderlined. The upstream and the downstream sequences are the humanβ-actin promoter and the SV40 polyA sequences in the pHβ-Apr1 neo vectorwith the addition of SalI and BamHI restriction enzyme sites. SEB andClone #2 Coding Sequence Table

[0855] Purified DNA templates from bacteria and human cells are preparedfor introduction of plasmid into human and bacterial cells by additionalmethods given in Ausubel F et al., supra. The plasmid DNA is grown up inE. coli in ampicillin containing LB medium. The cells were then pelletedby spinning a 5000 rpm for 10 min. at 5000 rpm., resuspended in cold TEpH 8.0, centrifuged again for 10 minutes. at 5000 rpm., resuspended in asolution of 50 mM glucose, 25 mM Tris-Cl pH 8.0, 10 mM EDTA and 40 mg/mllysozyme. After incubation for 5-10 min. with occasional inversion, 0.2N NaOH containing 1% SDS was added, followed after 10 minutes at 0° C.with 3 M potassium acetate and 2 M acetic acid. After 10 more minutes,the material was again centrifuged a 6000 rpm, and the supernatant wasremoved with a pipet. The pellet was then mixed into 0.6 vol.isopropanol (−20° C.), mixed, and stored at −20° C. for 15 minutes. Thematerial was then centrifuged again at 10,000 rpm for 20 min., this timein an HB4 singing bucket rotor apparatus after which the supernatant wasremoved and the pellet was washed in 70% EtOH and dried at roomtemperature. Next, the pellet was resuspended in 3.5 ml TE, followed byaddition of 3.4 g CsCl and 350 l of 5 mg/ml EtBr. The resulting materialwas placed in a quick seal tube, filled to the top with mineral oil. Thetube was spun for 3.5 hours at 80,000 rpm in a VTi80 centrifuge. Theband was removed and the material was centrifuged again making up thevolume with 0.95 g CsCl/ml and 0.1 ml or 5 mg/ml EtBr/ml in TE. The EtBrwas then extracted with an equal volume of TE saturated N-Butanol afteradding 3 volumes of TE to the band. Next, 2.5 vol. EtOH was added, andthe material was precipitated at −20° C. for 2 hours. The resultant DNAprecipitate is used as a DNA template.

[0856] Transfection of B16F10 Melanoma Cells

[0857] G418 sensitivity: B16F10 melanoma cells (B16s) were first testedfor sensitivity to G418 which will be used as the selectable marker. At400 μg/mL G418, B16s did not survive, while 200 and 300 μg/mL allowedsome survival.

[0858] Lipofectamine was used to produce stably transfected B16 cells.The conditions for transfection were those described protocol providedby Life Technologies. B16s were plated at 4×105 cells/well in 6 wellplates, using Murine Complete Medium (MCM) described in Report 2. Cellswere cultured overnight. Optimal density is 50-80% confluent and isusually achieved by 18-24 after seeding at 1-3×105 cells/well. DNAsources consisted of SEB-G418 resistance containing vector, vector DNAwith G418 resistance gene only, and control DNA from PSK401 (no G418resistance marker). DNA concentrations were determined for the SEBcontaining and control vectors. DNA source A₂₆₀ DNA (μg/ml) SEB 0.090.45 Vector only 0.13 0.65 PSK 401 0.15 0.75

[0859] Lipofectamine solutions and DNA solutions were prepared in 12×75mm tubes, using OPTI-MEM (Life Technologiies 31985). DNA solutionscontained approximately 2 μg in 100 uL OPTI-MEM; the LIPOFECTAMINEReagent was diluted by adding 6 or 12 uL to OPTI-MEM at a final volumeof 100 uL. The solutions were mixed and held at room temperature for 30minutes. Specific DNA and Lipofectamine conditions were as follows:

[0860] Plated cells were rinsed once with 2 ml/well OPTI-MEM. To theabove tubes, 0.8 mL OPTI-MEM. This mixture was then overlayed onto thewashed cell monolayers according to the above well designations. Cellswere incubated for 5 hours at 37° C. in 5% CO2. Murine Complete Mediumwith 20% FBS but no antibiotics was then added at 1 ml/well. Cultureswere refed with standard MCM, at 3 mL/well, after 24 hours. Three daysafter transfection, cells from each transfection condition weresubcultured by splitting the total cell suspension 90:10 into 150 mmplates (one plate received 90% of the cell suspension, the otherreceived the remaining 10%).

[0861] G418 Selection

[0862] All plates were refed at 6 days after transfection with mediumcontaining 400 μg/mL G418. Plates were refed every 2 to 3 days with G418containing medium until day 17 after transfection. No growth wasobserved in wells 1-4 as expected. Plates initiated with 90% of the cellsuspension and showing growth were harvested, frozen, and stored at −80°C.

[0863] Primary Subcloning

[0864] Ten colonies were selected from each well for wells 5, 7, 9, and11. Subcloning was accomplished by the use of cloning cylinders asfollows: After seating the cylinder, medium was aspirated and theisolated colony was washed once with 100 uL of warmed trypsin-EDTA. Thiswas aspirated and replaced with fresh tyrpsin-EDTA. After incubation at37° C. for 2 minutes, the cells were recovered by trituration andtransferred to a tube containing 1 ml MCM, then replated by addition of20 uL of cell suspension to 15 mL MCM with G418 in 150 mm plates. Theremaining cell suspension was plated into 24 well plates, 4 wells/cloneand all plates were maintained at 37° C., 5% CO2. The 6 well plates wereused to assess SEB expression on the cell surface as described underDetection of positive clones.

[0865] Secondary and Tertiary Subcloning and Preparation of FrozenStocks

[0866] These and all subsequent procedures were performed by me.Secondary subcloning was performed as above at 7 days after initiationof primary subclones. One colony/plate was selected for furthersubcloning (a total of 40 colonies) The cell suspension was prepared ina total volume of 1 mL; 100 uL was replated into 100 mm platescontaining 10 mL MCM with G418. The remaining cell suspension was platedin 96 well plates at 100/well, 2 replicates for assay. The 96 well platewas used for detection of intracellular expression of SEB describedunder Detection of positive clones.

[0867] Primary subcloning plates were cultured one additional day, thenharvested, frozen, and stored at −80° C. These frozen stocks aredesignated primary subclones. Secondary subclones were refed after 4days. Of 40 secondary clones, 36 regrew. Tertiary subcloning wasperformed after 8 days and frozen stocks of secondary clones wereprepared after 9 days. Tertiary clones were refed after 3 days inculture and subcultured after 7 days in culture. Plates were harvested,cells were resuspended in a total of 1 mL, and replated by addition of100 uL of the cell suspension to 100 mm plates with 15 mL MCM or 100μl/well in a 96 well plate. Frozen stocks of tertiary clones wereprepared.

[0868] Generation of Conditioned Medium for Assay of Supernatants

[0869] After 7 days, 100 mm plates of tertiary clones were againreplated. This time, cell counts were performed and 4.5×10⁵ cells wereplated in 12 well plates, one well/clone. The remaining cell suspensionwas frozen and stored at −80° C. After 4 days in culture, supernatantswere harvested, stored at 4° C., and the cells were replated into 100 mmplates. Supernatants were obtained from the 100 mm plates after 7 daysin culture. See Detection of positive clones. Frozen stocks were alsogenerated from these plates.

[0870] Development of ELISA with HRP Rabbit anti-SEB.

[0871] Final ELISA conditions were as follows:

[0872] Assay Plate ProBind (Falcon #3915)

[0873] Capture Antibody Rabbit anti-SEB (Toxin Technologies #LB1202), 10μg/mL in PBS, 50 μl/well, 1 hr, RT

[0874] Wash 3× with 0.1% casein, 0.1% Tween 20 in PBS

[0875] Blocking 1% casein in PBS, 250 μl/well, overnight, 4° C.

[0876] Antigen Supernatant used neat or SEB diluted in PBS, 50 μl/well,2 hr, RT

[0877] Wash As above

[0878] Primary Ab HRP Rabbit anti-SEB (Toxin Technologies #LBC202),1/300 in block buffer, 50 μl/well, 2 hr, RT

[0879] Substrate OPD, 2.5 mg/mL in citrate buffer, pH 5.0, 0.03% H₂0₂,100 μl/well, 15 min, RT

[0880] Stop 4M H₂SO₄, 100 μl/well

[0881] Read-out Absorbance at 490 nm (OD 490)

[0882] Results: SEB produced a dose response curve (linear range 60fg-60 pg/mL) and the background was very low. Vector only clonesproduced only background signals. One SEB transfected clone produced astrong signal, three produced moderate signals, and one other produced aweak but definite signal. Absorbance at 490 nm SEB+ Vector only 1 2 mean1 2 mean 9.1 0.097 0.112 0.104 0.079 0.102 0.091 9.2 0.127 0.123 0.1250.081 0.076 0.078 9.3 0.109 0.104 0.106 0.087 0.070 0.079 9.4 0.4440.393 0.418 0.077 0.077 0.077 9.5 0.163 0.087 0.125 0.075 0.074 0.0749.6 0.516 0.522 0.519 0.066 0.064 0.065 9.7 0.087 0.091 0.089 0.0960.084 0.090 9.8 0.386 0.450 0.418 0.080 0.071 0.075 9.9 0.137 0.1220.130 0.071 0.070 0.071 11.1 0.083 0.075 0.079 0.068 0.078 0.073 11.21.847 1.802 1.824 0.063 0.076 0.070 11.3 0.071 0.077 0.074 0.076 0.0740.075 11.4 0.087 0.084 0.086 0.083 0.085 0.084 11.5 0.161 0.220 0.1910.092 0.086 0.089 11.8 0.221 0.100 0.160 0.080 0.081 0.080 11.9 0.0800.091 0.085 0.077 0.072 0.074 11.10 0.290 0.254 0.272 0.081 0.112 0.09711.10 0.268 0.263 0.265 0.093 0.114 0.103

[0883] Based on the SEB standard curve, the following concentrationswere derived. Clone number(pg/ml) SEB 11.2 4.146 9.6 0.152 9.4 0.118 9.80.118 11.10 0.081

[0884] Cells are transfected ex vivo or in vivo and implanted in acancer-bearing host. These transfected cells are also used to stimulatehost lymphocytes ex vivo. Once activated, the lymphocytes areadministered to the host. The ex vivo or in vitro introduction of DNAinto cells is accomplished by methods that (1) form DNA precipitateswhich are internalized by the target cell; (2) create DNA-containingcomplexes with charge characteristics that are compatible with DNAuptake by a target cell; or (3) result in the transient formation ofpores in the plasma membrane of a target cell exposed to an electricpulse (these pores are of sufficient size to allow DNA to enter thetarget cell).

[0885] Generally, two factors determine the method used: the duration ofexpression required (i.e., transient versus stable expression) and thetype of cell to be transfected. The specific details of exemplaryprocedures are described herein. Transfections are carried out by wellestablished methods including calcium phosphate precipitations, DEAEDextran transfection, and electroporation.

[0886] Calcium Phosphate Precipitation

[0887] A commonly used ex vivo and in vitro method to transfer DNA intorecipient cells involves the co-precipitation of the DNA of interestwith calcium phosphate. With this technique, DNA enters the cell insufficient quantities such that the treated cells are transformed withrelatively high frequency. Using a variety of cell types, transfectionefficiencies of up to 10-3 have been obtained. This is the method ofchoice for the generation of stable transfectants.

[0888] Variations of the basic technique have been developed. If thetransfection involves the transfer of plasmid DNA, then high molecularweight genomic DNA isolated from a defined cell or tissue source can beincluded. The addition of such DNA, called carrier DNA, often increasesthe efficiency of transfection by the plasmid DNA. Upon arrival of theplasmid DNA/carrier DNA/calcium phosphate co-precipitate to the nucleusof the treated cell, the plasmid DNA integrates into the carrier DNA,often in the tandem array, and this assembly of plasmid and carrier DNA,called a transgenome, subsequently integrates into the chromosome of thehost cell.

[0889] Another procedural option is the addition of a chemical shockstep to the transfection protocol. Either dimethylsulfoxide or glycerolare appropriate. The optimal concentrations and lengths of treatmentvary according to cell type. The use of these agents dramatically affectcell viability and can be optimized as described elsewhere [Chen andOkayama, Mol. Cell. Biol. 7:2745 (1987)]. Specifically, incubation ofcells with the co-precipitate is optimal at 35° C. in 2-4% CO2 for 15-24hours. In addition, circular DNA is more active than linear DNA and afiner precipitate is obtained when the DNA concentration is between20-30 mg/ml in the precipitation mix.

[0890] It is noted that incubator temperature, CO2 concentration, andDNA concentration can be varied to obtain the desired result. Inaddition, the temperature and CO2 concentrations described below are notoptimal for cell growth and should be maintained only temporarily.

[0891] Method

[0892] Day 1: 1.3×106 cells are seeded per 100-mm dish. Cells are about75% confluent when used to seed the dishes.

[0893] Day 2: A large calcium phosphate cocktail mixture to transfectmany plates simultaneously is prepared. This protocol is given for 1 ml(or 1×100-mm dish equivalent) of solution. These amounts are scaled upas necessary, allowing for an appropriate amount of sample-transfererrors. Adherence to sterile technique is critical. Sterile reagents,tips, and tubes are used.

[0894] 1. Add 1-20 g DNA (1 mg/ml in sterile TE, 10 mM Tris-HCl 1 mMEDTA pH 7.05) to 0.45 ml sterile H2O. Note: First “sterilize” DNA byethanol precipitation with NaCl (0.1M final aqueous concentration) and2× volume 200% ethanol.

[0895] 2. Add 0.5 ml 2× HEPES buffered saline. Mix well.

[0896] 3. Add 50 ml of 2.5 M CaCl2, vortex immediately.

[0897] 4. Allow the DNA mixture to sit undisturbed for 15-30 minutes atroom temperature.

[0898] 5. Add 1 ml of the DNA transfection cocktail directly to themedium in the 100-mm dish (plated with cells on day 1).

[0899] 6. Incubate the dishes containing the DNA precipitate for 16hours at 37° C. Remove the media containing the precipitate and addfresh complete growth media.

[0900] 7. Allow the cells to incubate for 24 hours. Post-incubation, thecultures can be split for subsequent selection. Split cultures 1:5;however, to isolate individual colonies for further analysis, splitcultures 1:10 and 1:100.

[0901] DEAE Dextran Transfection

[0902] Typically, DEAE dextran transfection is used to transientlytransfect cells in culture. This method is highly efficient and theDNA/DEAE dextran mixture used for transfection is relatively easy toprepare. For example, this method yields transfection efficiencies of ashigh as 80 percent. DNA introduced into cells with this method, however,appears to undergo mutations at a higher rate than that observed withcalcium phosphate-mediated transfection.

[0903] Method

[0904] Briefly, a DEAE dextran mixture is prepared and the DNA sample ofinterest is added, mixed, and then transferred to the cells in culture.

[0905] Day 1: Cells are seeded at a concentration of 2×10⁴ cells/cm2 ina total volume of 2 ml/well (1.92×10⁵ cells/well of a six-well clusterdish). Cells should be about 75% confluent when used to seed the dishes.

[0906] Day 2: Resuspend 0.5 ml DEAE Dextran in Tris-buffered saline(TBS).

[0907] Final DEAE Dextran concentration should be about 0.04%. Observecell monolayers microscopically. Cells should appear about 60-70%confluent and well distributed. Bring all reagents to room temperature.Aspirate off growth media and wash monolayer once with 3 ml of phosphatebuffered saline (PBS), followed by one wash with 3 ml of TBS. Aspirateoff TBS solution and add 100-125 ml of the appropriateDNA/DEAE-Dextran/TBS mixture to the wells. Incubate dishes at roomtemperature inside a laminar flow hood. Rock the dishes every 5 minutesfor 1 hour, making sure the DNA solution covers the cells. After the1-hour incubation period, aspirate off the DNA solution and wash oncewith 3 ml of TBS followed by 3 ml of PBS. Remove the PBS solution byaspiration and replace with 2 ml of complete growth media containing 100M chloroquine. Incubate the dishes in an incubator set at 37° C. and 5%CO2 for 4 hours. Remove the media containing chloroquine and replacewith 2-3 ml of complete growth media (no chloroquine). Incubate thetransfected cells for 1-3 days, after which the cells will be ready foranalysis. The exact incubation period depends on the intent of thetransfection. Optimal expression typically occurs at 3dayspost-transfection.

[0908] Electroporation

[0909] Electroporation is a process whereby cells in suspension aremixed with the DNA to be transferred. This cell/DNA mixture issubsequently exposed to a high-voltage electric field. This createspores in the membranes of treated cells that are large enough to allowthe passage of macromolecules such as DNA into the cells. Such DNAmolecules are ultimately transported to the nucleus and a subset ofthese molecules are integrated into the host genome. The reclosing ofthe membrane pores is both time and temperature dependent and thus isdelayed by incubation at 0° C., thereby increasing the probability thatthe molecule of interest will enter the cell.

[0910] Electroporation appears to work on virtually every cell type.With this technique, the efficiency of nucleic acid transfer is high forboth transient transfection and stable transfection. One importanttechnical difference between electroporation and other competingtechnologies is that the number of input cells required forelectroporation is considerably higher.

[0911] Method

[0912] 1. Harvest exponentially growing cells such as tumor cells oraccessory cells by trypsinization, pellet, and wash twice withelectroporation buffer (Kriegler, M. Gene Transfer and Expression, W. H.Freeman and Co., New York, N.Y. (1991)).

[0913] 2. Resuspend cells in electroporation buffer at a concentrationof 2-20×10⁶ cells/ml in an electroporation cuvette.

[0914] 3. Add 5-25 mg of DNA that has been linearized to the cellsuspension

[0915] 4. Insert or connect the electroporation electrode according tothe manufacturer's instructions and subject cell/DNA mixture to anelectric field (pulse).

[0916] 5. Return cell/DNA mixture to ice and incubate for 5 minutes.

[0917] 6. Plate cells in non-selective medium. Biochemical selection maybe carried out 24-48 hours later.

[0918] Lipofectamine

[0919] In vitro cell transfections can be done in 12-well plates, using3.0 g plasmid DNA and Lipofectamine (GIBCO BRL), at 37° C. for 4 hours.After transfection, the cells are cultured in 2.0 ml complete medium for48 hours and the cells are harvested. The cells are then washed in PBS.Stably transfected Chinese hamster ovary (CHO) and B16 lines areisolated by selection in 1.0 mg/ml G418 (GIBCO BRL). Cells are grown andpassaged in medium containing G418 for 3-4 weeks Mock transfected celllines (cells transfected with vector only) are used as controls.

[0920] Viral Vectors

[0921] Recombinant viral vectors containing the nucleic acid of interestcan also be used to introduce nucleic acid into a cell ex vivo or invitro. It is noted that viral vectors are also used to transfect cellsin vivo. These viral vectors can be DNA viruses such as herpes viruses,adenoviruses, and vaccinia viruses or RNA viruses such as retroviruses.The method and materials required to produce and use these viral vectorsex vivo, in vitro, and in vivo are commonly known in the art and areused in the invention described herein (Sambrook, J. et al., supra).

[0922] Selection

[0923] Regardless of the method used to transfect a particular celltype, stably transfected cells are identified as follows. The DNA ofinterest contains a selectable marker. Typically, a selectable markerencodes a polypeptide that confers drug resistance and the DNAcontaining this resistance conferring nucleic acid is transfected intothe recipient cell. Post transfection, the treated cells are allowed togrow for a period of time (24-48) hours to allow for efficientexpression of the selectable marker. After an appropriate incubationtime, transfected cells are treated with media containing theconcentration of drug appropriate for the selective survival andexpansion of the transfected and now drug resistant cells.

[0924] Many drug as well as non-drug selection methods are known in theart and can be used in the invention described herein. For example, adetailed description of currently available drug selection strategies isprovided in Kriegler M., Gene Transfer and Expression, A LaboratoryManual, W. H. Freeman and Co. New York, N.Y. pp.103-107 (1991).

[0925] General Method

[0926] Sixteen hours after transfection, the transfected/infected cellsare fed with fresh, non-selective media. Twenty-four to forty-eighthours later, the cultures are split to a 1:5 or greater dilution andplated in drug-containing media. It is noted that cells are not placedin drug-containing media immediately after transfection in order toallow a sufficient amount of time for the drug resistance nucleic acidto be expressed and thus confer the drug resistant phenotype. Cellcultures are re-fed with drug-containing media every three days, atwhich time cultures are examined under a microscope to determine theefficiency of drug selection.

[0927] Site-Directed Mutagenesis by Polymerase Chain Reaction:Introduction of Restriction Endonuclease Sites by PCR

[0928] PCR is the preferred method for introducing any desired sequencechange into the DNA. The basic protocol is shown below. Material usedare: DNA sample to be mutagenized, Mineral oil pUC19 plasmid b vector orsimilar high-copy number plasmid having M13 flanking primer 500 ng/ml(100 pM/ml) flanking sequence primers incorporating the restrictionenzyme site TE buffer Chloroform 10× amplification buffer Bufferedphenol 2 mM 4dNTP mix 100% ethanol 500 ng/ml (100 pM/ml) M13 Appropriaterestriction flanking sequence primers: endonucleases forward (NEB) andreverse (NEB) 5 U/ml Taq DNA polymerase 500 ml microcentrifuge tube

[0929] The process steps are as follows:

[0930] 1. Subclone DNA to be mutagenized into high-copy number vectorusing restriction sites flanking the area to be mutated.

[0931] 2. Prepare template DNA by plasmid miniprep. Resuspend 100 ng inTE buffer to 1 ng/ml final.

[0932] 3. Synthesize oligonucleotide primers and purify by denaturingpolyacrylamide gel electrophoresis. Resuspend oligonucleotides in 500 lTE buffer. Determine absorbance at A₂₆₀ and adjust to 500 ng/ml.

[0933] 4. Combine the following in each of two 500 1 microcentrifugetubes, adding oligonucleotides 1 and 2 to separate tubes:

[0934] 10 ml (10 ng) template DNA

[0935] 10 ml 10× amplification buffer

[0936] 10 ml 2 mM 4dNTP mix

[0937] 1 ml (500 ng) oligonucleotide 1 or 2 (100 pM final)

[0938] 1 ml (500 ng) appropriate M 13 flanking sequence primer, forwardor reverse (100 pM final).

[0939] H₂O to 99.5 ml

[0940] 0.5 ml Taq DNA polymerase (5 U/ml)

[0941] Overlay reaction with 100 ml mineral oil.

[0942] 5. Carry out PCR in an automated thermal cycler for 20 to 25cycles under the following conditions: 45 sec—93° C.; 2 min—50° C.; 2min—72° C. After last cycle, extend for an additional 10 min at 72° C.

[0943] 6. Analyze 4 l by nondenaturing agarose or occurrence gelelectrophoresis to verify that the amplification has yielded thepredicted product.

[0944] 7. Remove mineral oil and extract once with chloroform to removeremaining oil. Extract with buffered phenol and concentrate byprecipitation with 100% ethanol.

[0945] 8. Digest half the amplified DNA with the restrictionendonucleases for the flanking and introduced sites. Purify digestedfragments on a low gelling/melting agarose gel.

[0946] 9. Ligate and subclone both fragments into an appropriatelydigested vector to obtain a recombinant plasmid containing a single DNAfragment incorporating the new restriction site.

[0947] 10. Transform plasmid into E. coli. Prepare DNA by plasmidminiprep.

[0948] 11. Analyze amplified fragment portion of plasmid by DNAsequencing to confirm the addition of the mutation.

[0949] Introduction of Point Mutation by PCR

[0950] Materials

[0951] DNA sample to be mutagenized

[0952] Oligonucleotide primers incorporating the point mutation

[0953] Klenow fragment of E. coli DNA polymerase I

[0954] Appropriate restriction endonuclease

[0955] Procedural Steps:

[0956] 1. Prepare template DNA (steps 1 and 2 of Basic Protocol).

[0957] 2. Synthesize and purify oligonucleotide primers (3 and 4).

[0958] 3. Amplify template DNA (steps 4 and 5 of Basic Protocol 1).After final extension step, add 5 U Klenow fragment and incubate 15 minat 30° C.).

[0959] 4. Analyze and process reaction (steps 6 and 7 of BasicProtocol).

[0960] 5. Digest half the amplified fragments with the restrictionendonucleases for the flanking sequences. Purify digested fragments on alow gelling/melting agarose gel.

[0961] 6. Subclone the two amplified fragments into an appropriatelydigested vector by blunt-end ligation.

[0962] 7. Carry out steps 10 and 11 of Basic Protocol.

[0963] Introduction of a Point Mutation by Sequential PCR

[0964] Procedural Steps:

[0965] 1. Prepare the template DNA (steps 1 and 2 of Basic Protocol 1).

[0966] 2. Synthesize and purify the oligosaccharide primers (5 and 6).

[0967] 3. Amplify the template and generate blunt-end fragments (step 3of Basic Protocol).

[0968] 4. Purify fragments by nondenaturing agarose gel electrophoresis.Resuspend in TE buffer at 1 ng/ml.

[0969] 5. Combine the following in 500 ml microcentrifuge tube andoverlay with 100 ml mineral oil:

[0970] 10 ml (10 ng) each amplified fragment

[0971] 1 ml (500 ng) each flanking sequence primer (each 100 pM final)

[0972] 10 ml 10× amplification buffer

[0973] 10 ml 2 mM 4dNTP mix

[0974] 0.5 ml Taq DNA polymerase (5 U/ml)

[0975] 6. Carry out PCR for 20 to 25 cycles (step 5 of Basic Protocol1). Analyze and process the reaction mix (steps 6 and 7 of BasicProtocol 1).

[0976] 7. Digest cDNA fragment with appropriate restriction endonucleasefor the flanking sites. Purify fragment on a low gelling/melting agarosegel. Subclone into an appropriately digested vector.

[0977] 8. Carry out steps 10 and 11, Basic Protocol 1.

[0978] Genomic Targeting and Genetic Conversion in Cancer Therapy

[0979] A number of cellular transformations are due, in large part, to asingle base mutation that alters the function of the expressed protein.Alterations in the DNA sequence of a gene involved in cell proliferationcan have a significant effect on the viability of particular cells.Thus, the capacity to modulate the base sequence of such a gene would bea useful tool for cancer therapeutics. An experimental strategy thatcenters around site-specific DNA base mutation or correction using aunique chimeric oligonucleotide has been developed. This chimericmolecule has demonstrated higher recombinogenic activities thanidentical oligonucleotides containing only DNA residues, both in vitroand in vivo. The chimeric molecule is designed to hybridize to a targetsite within the genome and induce a single base mismatch at the residuetargeted for mutation. The DNA structure created at this site isrecognized by the host cell's repair system which mediates thecorrection reaction. For example, the bcr-abl fusion gene, the productof a translocation between human chromosomes 9 and 22, and the cause ofchronic myelogenous leukemia (CML) can be targeted for gene correction.Fusion genes or mutations which abound in cancer cells are excellenttargets for correction especially if (1) they are unique and arerecognized by the immune system as dominant or subdominant epitopes, (2)they are a single copy target; (3) the DNA sequence of the fusion geneor mutation is unique. The goal of such experiments is to knock-out thefusion gene by changing an amino acid codon into a stop codon through achimeric directed DNA repair system.

[0980] Targeted Gene Correction of Episomal DNA in Mammalian CellsMediated by a Chimeric RNA/DNA Oligonucleotide

[0981] An experimental strategy to facilitate correction of single-basemutations of episomal targets in mammalian cells has been developed. Themethod utilizes a chimeric oligonucleotide composed of a contiguousstretch of RNA and DNA residues in a duplex conformation with doublehairpin caps on the ends. The RNA/DNA sequence is designed to align withthe sequence of the mutant locus and to contain the desired nucleotidechange. Activity of the chimeric molecule in targeted correction is usedin a with the aim of correcting a point mutation in the gene encodingthe human liver/bone/kidney alkaline phosphatase. When the chimericmolecule is introduced into cells containing the mutant gene on anextrachromosomal plasmid, correction of the point mutation isaccomplished with a frequency approaching 30%. These results extend theusefulness of the oligonucleotide-based gene targeting approaches byincreasing specific targeting frequency.

[0982] The site directed mutagenesis is used to carry out using thechimeric DNA/RNA structure which enables the construct to target tumorcells in vivo and in vitro. Such targeting structures include targetseeking moieties and can in principle be any structure that is able tobind to a cell surface structure or that binds via biospecific affinity.The target seeking moiety is primarily a disease specific structureselected among hormones, antibodies, growth factors. The biospecificaffinity counterpart may include interleukins (especially interleukin-2)antibodies (full length antibody, Fab, F(ab)′₂, Fv, single chainantibody and any other antigen binding antibody fragments (such as Fab)directed to a cells surface epitope or more preferably towards thebinding epitope for the a specific antibody. They may also includepolypeptides binding to the constant domains of immunoglobulins (e.g.,protein A and G and L), lectins, streptavidin, biotin etc.

[0983] The term antibodies comprises monoclonal as well as polyclonalpreparations. The targeting moiety may also be directed toward uniquestructures on more or less healthy cells that regulate or control thedevelopment of a disease or ligands for specific receptors on tumorcells). The targeting structure may be a nucleic acid, lipid orcarbohydrate and variations thereof which target receptors on thediseased cell. The targeting is not confined to diseased cells but mayinclude additional normal cells as well.

[0984] Synthesis and Purification of Oligonucleotides.

[0985] The chimeric oligonucleotides are synthesized on a 0.2-mol scaleby using the 1000 Å-wide-pore CPG on the ABI 394 DNA/RNA synthesizer.The exocyclic amine groups of DNA phosphoramidites (Applied Biosystems)are protected with benzoyl for adenosine and cytidine and isobutyryl forguanosine. The 2′-O-methyl RNA phosphoramidites (Glen Research,Sterling, Va.) are protected with a phenoxyacetyl group for adenosine,dimethylformamide for guanosine and an isobutyryl group for cytidine.After the synthesis is complete, the base-protecting groups are removedby heating in ethanol/concentrated ammonium hydroxide, 1:3 (vol/vol),for 20 h at 55° C. The crude oligonucleotides are purified bypolyacrylamide gel electrophoresis. The entire oligonucleotide sample ismixed with 7 M urea/10% (vol/vol) glycerol. heated to 70° C., and loadedon a 10% polyacrylamide gel containing 7 M urea. After gelelectrophoresis, DNA is visualized by UV shadowing, dissected from thegel, crushed, and eluted overnight in TE buffer (10 mM Tris-HCl/1 mMEDTA, pH 7.5) with shaking. The eluent containing gel pieces arecentrifuged through 0.45-um (pore size) spin filter (Millipore) andprecipitated with ethanol. Samples are further desalted with a G-25 spincolumn (Boerhinger Mannheim) and greater than 95% of the purifiedoligonucleotides are found to be full length.

[0986] Transient Transfection and Measurements of Activity

[0987] CHO cells were maintained in Dulbecco's modified Eagle's medium(DMEM) (BRL) containing 10% (vol/vol) fetal bovine serum (FBS; BRL).Transient transfection is carried out by addition of 10 g of the plasmidwith 10 g of Lipofectin in 1 ml of Optimem (BRL) to 2×10⁵CHO cells in a6-well plate. After 6 h. various amounts of oligonucleotide is mixedwith 10 g of Lipofectin in 1 ml of Optimem and added to each well. After18 h, the medium is aspirated and 2 ml of DMEM containing 10% FBS wasadded to each well. Histochemical staining was carried out (19), 24 hafter transfection of the oligonucleotide. Spectrophotometricmeasurements are carried out by the ELISA amplification system (BRL).Transfection is carried out in triplicate in a 96-well plate. Theamounts of reagents and cells are 10% of that used for the 6-well plate.Cells were washed three times with 0.15M NaCl and lysed in 100 ml ofbuffer containing 10 mM NaCl, 0.5 Nonidet P-40, 3 mM MgCl2, and 10 mMTris-HCl (pH 7.5), 24 h after transfection with chimericoligonucleotides. A fraction of cell lysates (20 ml) incubated with 50 lof ELISA substrate and 50 ml of ELISA amplifier (BRL), the reaction isstopped by addition of 50 ml of 0.3 M H2S04 after 5 min of incubationwith amplifier. The extent of reaction is carried out within the linearrange of the detection method. The absorbance is read by an ELISA platereader (BRL) at a wavelength of 490 nm.

[0988] Hirt DNA Isolation, Colony Hybridization, and Direct DNASequencing of PCR Fragments

[0989] The cells are harvested for vector DNA isolation by a modifiedalkaline lysis procedure, 24 h after transfection with the chimericoligonucleotide. Hirt DNA is transformed into Escherichia coli DH5acells (BRL). Colonies from Hirt DNA are screened for specifichybridization for each probe designed to distinguish the point mutation.Colonies were grown on ampicillin plates, lifted onto nitrocellulosefilter paper in duplicates, and processed for colony hybridization. Theblots were hybridized to ³²P-end-labeled oligonucleotide probes at 37°C. in a solution containing 5× Denhardt's solution, 1% SDS, 2×SSC, anddenatured salmon sperm DNA (100 mg/ml). Blots were washed at 52° C. inTMAC solution (3.0 M teramethylammonium chloride/50 mM Tris-HCl, pH8.0/2 mM EDTA/0.1% SDS). Plasmid DNA was made from 20 colonies shown tohybridize to either of the probes by using the Qiagen miniprep kit(Chatsworth. Calif.). Several hundred bases flanking key positions ofeach plasmid are sequenced in both directions by automatic sequencing(ABI 373A, Applied Biosystems). A 192-bp PCR-amplified fragment aregenerated by Vent polymerase (New England Biolabs. Mass.), utilizingprimers corresponding to positions of the known cDNA flanking position.The fragment is gel-purified and subjected to automatic DNA sequencing(ABI 373A, Applied Biosystems).

[0990] Oligonucleotide Synthesis

[0991] Chimeric RNA/DNA oligonucleotides for both transcribed andnontranscribed factor IX were synthesized by Applied Biosystems, Inc.(Foster City, Calif.) as previously described. The oligonucleotides areprepared with DNA and 2-O-methyl RNA phosphoramidite nucleoside monomerson an ABI 394 DNA/RNA synthesizer, purified by HPLC and quantified by UVabsorbance. More than 95% of the purified oligonucleotides aredetermined to be full length.

[0992] Cell Isolation and Transfections

[0993] Cells are isolated, by a two-step collagenase perfusion aspreviously described. The purified cells are plated on Primaria plates(Becton Dickinson, Franklin Lakes, N.J.) at a density of 4×10⁶ cells per35-mm dish and maintained in supplemented William's E medium. Eighteenhours after plating, the cells are washed and transfected with thechimeric molecules complexed to polyethylenimine (PEI). A pH 7.0, 10 mMstock solution of PEI (800 kDa) (Fluka Chemical Corp., Ronkonkoma, N.Y.)is prepared. Briefly, the chimeric oligonucleotides are complexed with10 mM PEI at 9 equivalents of PEI nitrogen per chimeric phosphate in 100l of 0.15 M NaCl and transfected in 1 ml of medium at finalconcentrations of 150, 300 or 450 nM. PEI is lactosylated by couplinglactose to 30% of the nitrogen amines using sodium cyanoborohydride(Sigma Chemical Company, St. Louis, Mo.). Cells are also transfected 1with 100 l of 0.15 M NaCl containing the lactosylated 800-kDa and 25-kDaPEI chimeric complexes (Sigma) at final concentrations of 90, 180 or 270nM. After 18 h, an additional 2 ml of medium is added to the transfectedcultures for the remaining 6 or 30 h of incubation. Vehicle controltransfections utilize the same amount of PEI, but substituted an equalvolume of 10 mM Tris-HCl, pH 7.6, for the oligonucleotides.

[0994] DNA/RNA Isolation and Cloning

[0995] The cells were harvested by scraping 48 h after transfection.Genomic DNA larger than 100-150 base pairs was isolated using the highlypure PCR template preparation kit (Boehringer Mannheim, Indianapolis,Ind.). RNA was isolated using RNAzoI 8 (Tel-Test, Inc., Friendswood,Tex.), according to the manufacturer's protocol. PCR amplification of afragment of the gene in question gene is performed with 300 ng of theisolated DNA from either the primary cell culture.

[0996] The primers were designed (Oligos Etc., Wilsonville, Oreg.)corresponding to nucleotides to cDNA to be corrected. Primer annealingis carried out at 59° C., and the samples are amplified for 30 cyclesusing Expand Hi-fidelity polymerase (Boehringer Mannheim). To rule outPCR artifacts, 300 ng of control DNA is incubated with 0.5, 1.0 and 1.5g of the oligonucleotide before the PCR-amplification reaction.Additionally, 1.0 g of the chimeric alone is used as the “template” forthe PCR amplification.

[0997] RT-PCR amplification is done utilizing the Titian one tube RT-PCRsystem (Boehringer Mannheim) according to the manufacturer's protocoland by using the same primers as those used for the DNA PCRamplification. To rule out DNA contamination, the RNA samples aretreated with RQ1 DNase-free RNase (Promega Corp., Madison, Wis.) andRT-PCR negative controls of RNased RNA samples were performed inparallel with the RT-PCR reaction. Each of the PCR reactions is ligatedinto the TA cloning vector pCR 2.1 (Invitrogen, San Diego, Calif.) andtransformed into frozen competent E. coli.

[0998] Nuclear Uptake of the Chimeric Molecules

[0999] Nuclear localization of fluorescently labeled chimericoligonucleotides was determined in the isolated cells. For in vivostudies, 250 l saline containing 75 g of fluorescently labeled chimericoligonucleotides complexed to PEI is injected directly into the exposedcaudate lobe. The animals are killed 24 h post injection, the tumortargeted is removed, bisected longitudinally, embedded using OCT andfrozen cryosections were cut ˜10 pm thick, fixed, processed and examinedusing a MRC1000 confocal microscope (Bio-Rad, Inc., Hercules, Calif.).

[1000] In vivo Delivery of the Chimeric Oligonucleotides

[1001] Vehicle controls and lactosylated 25-kDa PEI at a ratio of 6equivalents of PEI nitrogen per chimeric phosphate are prepared in 300 lof 5% dextrose. The aliquots are administered either as a single dose of100 g or divided doses of 150 g and 200 g on consecutive days. Five dayspost injection, tumor tissue is removed for DNA and RNA isolation. DNAis isolated. RNA is isolated for RT-PCR amplification of the same regionas the genomic DNA using RNAexol and RNAmate (Intermountain ScientificCorp., Kaysville, Utah) according to the manufacturer's protocol.

[1002] Colony Hybridization and Sequencing

[1003] Eighteen to 20 h after plating, the colonies were lifted onto MSIMagnaGraph nylon filters (Micron Separations, Inc., Westboro, Mass.),replicated and processed for hybridization according to themanufacturer's recommendation. The filters were hybridized for 24 h with32P-end-labeled oligonucleotide probes (Life Technologies, Inc.,Gaithersburg, Md.), where the underlined nucleotide is the target ofmutagenesis. Hybridizations are performed at 37° C., and the filters areprocessed following hybridization for autoradiography. Plasmid DNAisolated from colonies identified as hybridizing with the 32P-labeledprobes is subjected to automatic sequencing using the forward andreverse primers, as well as gene specific primer corresponding tonucleotides of the normal gene.

EXAMPLE 2 Cells Transfected with Nucleic Acids Encoding SAgs

[1004] Cultured VX-2 carcinoma cells were shown to retain theirtumorigenic activity after implantation into New Zealand white rabbits.Progressive tumor outgrowth was observed over a 3 week period. Nucleicacid encoding SEB isolated and characterized by Gaskill et al, J. Biol.Chem. 263:6276 (1988) and Ranelli et al., Proc. Natl Acad. Sci. USA82:5850 (1985) were used to transfect tissue cultured VX-2 carcinomacells using transfection methodology described in Example 1.Transfectants were selected using Q418 and the survival ofSEB-transfected VX-2 carcinoma cells was observed. In additionalexperiments, attempts were made to transfect murine 205 and 207 tumorcells with nucleic acid encoding SEB(the kind gift from Dr. Saleem Khan)and Streptococcal pyrogenic exotoxin A (the kind gift of Dr. JosephFerretti). Successful transfection of murine MCA 205 and B16 cells bynucleic acids encoding SEA and SEC2 was achieved shortly thereafter byintegrating the SAg DNA into several retroviral vectors (MFG NEO)containing a growth hormone leader sequence under the control of a chickβ-actin promoter (Krause J C et al., J. Hematotherapy 6: 41-51 (1997)).In addition, murine tumors MCA 205 fibrosarcoma cells and a spontaneousmammary carcinoma cells were successfully transfected with nucleic acidsencoding SEB (provided by Dr. Saleem Khan) using the β-actin promoter.Transfected mammary carcinoma cells induced T cell proliferation invitro. To demonstrate the anti-tumor capacity of tumor cells transfectedwith nucleic acid encoding a SAg, these transfectants were injected i.p.into syngeneic hosts with established mammary carcinomas. Thesetransfectants demonstrated a capacity to reduce micrometastases of wildtype mammary tumor in vivo assessed in a clonogenic lung metastasesassay. The anti-tumor effect produced by the SEB transfectants wasenhanced significantly by the co-administration of tumor cellstransfected with nucleic acids encoding the costimulating molecule B7-1.

EXAMPLE 3 Naked SAg DNA and Cells Co-transfected with SAg DNA and withAdditional Nucleic Acid Encoding Anti-Tumor Motifs or Products

[1005] Nucleic acids encoding a SAg are injected in naked or plasmidform into a host with cancer as a means of activating T cells andinitiating an anti-tumor response. They may also be used as a vaccine toprevent the occurrence or recurrence of tumor in a host. Undercircumstances where it is desirable to activate CD4 cells to produce aTH-1 cytokine response the nucleic acid construct used to transfectcells contains immunostimulatory sequences such as unmethylated CpGsequences. Nucleic acids encoding SAgs may be co transfected into tumorcells together with nucleic acid encoding other constituents capable ofpromoting an anti-tumor response. A list of possible components ofnucleic acid constructs for direct administration and/or transfection oftumor cells which are administered to the host is presented in Table 2.

[1006] The nucleic acid construct or constructs are administered to ahost intramuscularly, intradermally, systemically, parenterally,intratumorally, orally or locally (in the vicinity of the tumor).Alternatively, the construct is administered via a catheter or otherdevices known in the art into the tumor vasculature supplying all orpart of a tumor. When the construct is injected systemically, thenucleic acid construct is directed to the tumor using an anti-tumorantibody or ligand specific for a tumor receptor or receptor on thetumor neovasculature or stroma. The antibody or ligand or othertargeting structures are conjugated to the SAg nucleic acid construct inorder to facilitate the introduction of the construct into tumor cells.Nucleic acid/polypeptide complexes or nucleic acid/viral complexes areused to target a specific receptor on the tumor vasculature or stroma.

[1007] Chemical Conjugation of SAg Nucleic Acids to VTs,Apolipoproteins, HPV Epitopes or Other Polypeptides/Proteins Listed inTables I and II.

[1008] The following section describes actual physical conjugatesbetween poly- or oligonucleotides and peptides or proteins. SAg nucleicacid conjugates are prepared by chemical modification of nucleic acidsat specific sites within individual nucleotides or withinoligonucleotides such that a protein can be bound to a DNA or RNApolymer.

[1009] Derivatization may be accomplished through discrete sites on theavailable bases, sugars, or phosphate groups to create primary amines,sulfhydryls, carboxylates or phenolates. The chemical modification ofnucleic acids can encompass several strategies. The initialderivatization may be the addition of a spacer arm to a particularreactive group on the nucleotide structure. Such a spacer typicallycontains a terminal functional group, such as an amine, that can be usedto couple another molecule. The spacer may be used to react with across-linking agent, such as a heterobifunctional compound that canfacilitate the conjugation of a protein or another molecule to themodified nucleotide. TABLE II NUCLEIC ACID CONSTRUCTS AND CELLSSAg-encoding DNA is used alone or together with DNA encoding other cellsurface moieties useful in generating antitumor immunity. Genes or theirproducts are shown in column 1, source information is shown in column 3,preferred cells to be transformed, transfected or transduced with theDNA are shown in column 2. All of references are incorporated byreference in their entirety. Gene or Gene Product Cells transformedReference or Source 1. SAg Tumor [See Text] 2. Enterotoxin Tumor [SeeText] 3. SAg receptor Tumor [See Text] 4. Enterotoxin receptor Tumor[See Text] 5. CD1 receptor(s) Tumor Martin L H et al., Proc Natl. Acad.Sci. 83: 9154-9158 (1986) 6. CD14 receptor Tumor Ferrero, E et al., J.Immunol. 145: 331-336 (1990) 7. CD44 encoding nucleic acids T or NKTNottenburg, C et al. Proc. Natl. Acad. Sci. 66: 8521-8525 (1992) 8.Carbohydrate modifying enzymes Tumor, T or NKT Sheng, Y et al. Int. J.Cancer 73: 850-858 (1997) 9. TCR V chain Tumor Tillinghast, JP et al.,Science 233: 879-883 (1986) 10. Staph/Strep hyaluronidase Tumor Hynes WL et al., Infect. Immun., 63: 3015- 3020 (1995) 11. Staph/Streperythrogenic toxin Tumor McShan W M, et al., Adv. Exp. Med. Biol. 418:971-973 (1997) 12. Staphylococcal β -hemolysin Tumor Projan S J et al.,Nucleic Acid Res. 3305- 3309 (1989) 13. Strep capsular polysaccharideTumor Lin, WS et al., J. Bacteriol. 176: 7005-7016 (1994) 14. Staphstaphylocoagulase Tumor Kaida S. et al., J. Biochemistry 102: 1177- 1186(1987) 15. Staph Protein A Tumor Shuttleworth, H L et al., Gene 58:283-295 (1987) 16. Staph Protein A domain D Tumor Roben, P W et al., J.Immunol. 154: 6347- 6445 (1995) 17. Staph Protein A Domain B TumorGouda, H et al., Biochemistry, 31: 9665- 9672 (1992) 18.Immunostimulatory protein Tumor, T or NKT Tokunaga, T et al., Microbiol.Immunol. 36: 55-66, (1992) 19. Costimulatory protein Tumor Entage, PC etal., J. Immunol. 160: 2531- 2538 (1998) 20. SAg-mimicking nucleic acid Tor NKT 21. Glycophorin Tumor Siebert, P D. et al., Proc. Natl. Acad.Sci. USA 83 1665-1669 (1986) 22. Mannose receptor Tumor Kim S J. et al.,Genomics 14: 721-727 (1992) 23. Angiostatin Tumor Cao, Y. et al., J.Clin. Invest 101: 1055-1063 (1998) 24. Chemoattractant Tumor Ames, R S.et al., J. Biol. Chem. 271: 20231- 20234 (1996) 25. Chemokine TumorNagira, M et al., J. Biol. Chem. 272: 19518- 19524 (1997) 26.Transcription factor Tumor, T or NKT Schwab M et al., Mol. Cell Biol. 6:2752-2758 (1986) 27. Transcription factor-binding Tumor, T or NKTnucleic acid 28. SAg/peptide conjugate Tumor 29. Glyco-SAg Tumor 30.Staph. Global regulator gene agr Tumor Balaban, N. et al., Proc. Natl.Acad. Sci. USA 92: 1619-1623 (1995) 31. Lipid A biosynthetic genes IpxA-Tumor Schnaitman C A et al., Microbiological D Reviews 57: 655-682(1993) 32. Mycobacterial mycolic acid Tumor Fernandes N D et al., Gene170: 95-99 (1996); Mathur M et al., J. Biol. Chem. 267: 19388-19395(1992) 33. c-abl oncogene amplified in Tumor Scherle P A et al., Proc.Natl. Acad. Sci. USA chronic myel. Leukemia 87: 1908 (1990); HeisterkampN et. al., Nature 344: 251-253 (1990) 34. erbB2 (HER2/neu) oncogeneTumor Schechter A L et al., Science 229: 976 (1985); Bargmann C L Nature319: 22 (1986); Hung M C et al., Proc. Natl. Acad Sci. 83: 261 (1986);Yamamoto T et al., Nature 319: 230 (1986) 35. IGF-1 receptor gene TumorAbbott A M et al., J. Biol. Chem. 267: 10759- 10763 (1992); Scott J etal., Nature 317: 260-262 (1985); Liu J et al., Cell 75: 59-63 (1993) 36.VEGF Tumor Tischer E et al., J. Biol. Chem. 266: 11947- 11954 (1991) 37.Strep emm-like gene family Tumor Kehoe M A, In: Cell-Wall AssociatedProteins in Gram-Positive Bacteria in Bacterial Cell Wall, Ghuysen J Met al., eds, Elsevier, Amsterdam, 1994 38. iNOS Tumor Xie Q W et al.,Science 256: 225-228 (1992) 39. Apolipoproteins (e.g., Lp(a), Tumor [SeeText] apoB-100, apoB-48, apoE) 40. LDL & oxyLDL receptors (e.g., Tumor[See Text] LDL oxyLDL, acetyl-LDL, VLDL, LRP, CD36, SREC, LOX-1macrophage scavenger receptors) 41. Chemokine receptor Tumor/Accessorycells [See Text]

[1010] If enzymatic methods are used to incorporate a small spacer intoan oligonucleotide, subsequent chemical conjugation steps still areneeded to add the protein moiety. In some cases, if an oligonucleotidecontains the appropriate functional group, a protein may be directlycoupled using chemical methods. Many of the chemical derivatizationmethods employed in these strategies involve the use of an activationstep that produces a reactive intermediary. The activated species thencan be used to couple a molecule containing a nucleophile, typically aprimary amine.

[1011] A preferred method is to amidate the 5′ PO4 of theoligonucleotide with EDC and then couple cystamine to the 5′ amidatedoligonucleotide. EDC will add an amide to the oligonucleotide to form aphosphoramidate linkage. After the addition of cystamine the disulfideis reduced with an agent such as dithiothreitol (DTT) to produce a free5′ sulfhydryl. The derivatized oligonucleotide is then coupled to aprotein chain (e.g., a verotoxin A or B chain) that has been activatedwith a heterobifunctional cross-linker such as succinimidyl4(N-maleimidomethyl)cyclohexane 1-carboxylate (SMCC) which reacts withthe amines on the protein which then react with the sulfhydryls on thederivatized oligonucleotide. N-succinimidyl S-actylthioacetate (SATA) isuseful for adding a free thiol or sulfhydryl group to a molecule lackingthis moiety. With this modification, “protected” sulfhydryl is formedwhich may be stored indefinitely in this protected state.

[1012] When needed, the acetyl group on the protected sulfhydryl isremoved to reveal the sulfhydryl for conjugation to another molecule. Aheterobifunctional agent such as SMCC or N-Succinimidyl3-(2-pyridylthio)propionate (SPDP) may be directly added to the amidatedoligonucleotide phosphate group to produce a free sulfhydryl unit forreactivity with the protein or peptide.

[1013] Chemical Conjugation of Polypeptides/Proteins to SAg DNA viaCarbodiimide Reaction with the 5′-Phosphates (Phosphoramidate Formation)

[1014] The water-soluble carbodiimide EDC, rapidly reacts with acarboxylate or phosphate to form an active complex able to couple with aprimary amine-containing compound. The carbodiimide activates an alkylphosphate group to a highly reactive phosphodiester intermediate.Diamine spacer molecules or amine-containing peptides then may reactwith this active species to form a stable phosphoramidate bond.Alternatively, bis-hydrazide compounds may be coupled to DNA using thisprotocol to yield a terminal hydrazide functional group able to reactwith aldehyde-containing molecules (Ghosh et. al., 1989). These methodspermit specific labeling of SAg DNA only at the 5′ end.

[1015] The following protocol describes the modification of SAg DNA orRNA oligonucleotides at their 5′-phosphate ends with a bis-hydrazidecompound, such as adipic acid dihydrazide or carbohydrazide. A similarprocedure for coupling the diamine compound cystamine is describedbelow.

[1016] Protocol

[1017] 1. Weigh out 1.25 mg of the carbodiimide1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDC) intoa microfuge tube.

[1018] 2. Add 7.5 ml of SAg RNA or DNA that has 5′ phosphate groups. Theconcentration of the oligonucleotide should be 7.5-15 nmol or a total ofabout 57-115.5 mg. Also immediately add 5 ml of 0.25 M bis-hydrazidecompound dissolved in 0.1 M imidazole, pH 6.

[1019] 3. Mix (e.g., by vortexing) and centrifuge in a microfuge for 5min at maximal rpm.

[1020] 4. Add an additional 20 ml of 0.1 M imidazole, pH 6. Mix andallow to react for 30 mm at room temperature.

[1021] 5. Purify the hydrazide-labeled oligonucleotide by gel filtrationon Sephadex G-25 using 10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA,pH 7.2. The oligonucleotide now may be conjugated with analdehyde-containing molecule.

[1022] Sulfhydryl Modification of SAg DNA

[1023] Creating a sulfhydryl group on SAg DNA allows conjugationreactions to be done with sulfhydryl-reactive heterobifunctionalcross-linkers providing increased control over the derivatizationprocess. Proteins are activated with a cross-linking agent containing anamine-reactive and a sulfhydryl-reactive end, such as SPDP, leaving thesulfhydryl-reactive portion free to couple with the modified DNAmolecule. Having a sulfhydryl group on the SAg DNA directs the couplingreaction to discrete sites on the nucleotide strand, thus betterpreserving hybridization ability in the final conjugate. In addition,heterobifunctional cross-linkers of this type allow two- or three-stepconjugation procedures which result in better yield of the desiredconjugate than do homobifunctional reagents.

[1024] Cystamine Modification of 5′ Phosphate Groups on SuperantigenNucleotides Using EDC

[1025] SAg DNA or RNA is modified with cystamine at the 5′ phosphategroups using the carbodiimide reaction described above. In someprocedures, the reaction is carried out in a two-step process by firstforming a reactive phosphorylimidazolide by EDC conjugation in animidazole buffer. Next, cystamine is reacted with the activatedoligonucleotide, causing the imidazole to be replaced by the amine andcreating a phosphoramidate linkage. Reduction of the cystamine-labeledoligonucleotide using a disulfide reducing agent releases2-mercaptoethylamine and creates a thiol group.

[1026] Protocol

[1027] 1. Weigh out 1.25 mg of the carbodiimide1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride (EDC) intoa microfuge tube.

[1028] 2. Add 7.5 ml of SAg RNA or DNA that has 5′ phosphate groups. Theconcentration of the oligonucleotide should be 7.5-15 nmol or a total ofabout 57-115.5 mg. Also immediately add 5 ml of 0.25 M cystamine in 0.1M imidazole, pH 6.

[1029] 3. Mix (e.g., by vortexing) and centrifuge in a microfuge for 5min at maximal rpm.

[1030] 4. Add an additional 20 ml of 0.1 M imidazole, pH 6. Mix andallow to react for 30 mm at room temperature.

[1031] 5. For reduction of the cystamine disulfides, add 20 ml of 1 MDTT and incubate at room temperature for 15 mm. This will release2-mercaptoethylamine from the cystamine modification site and create thefree sulfhydryl on the 5′ terminus of the oligonucleotide.

[1032] 6. Purify the SH-labeled oligo by gel filtration on Sephadex G-25using 10 mM sodium phosphate, 0.15 M NaCl, 10 mM EDTA, pH 7.2. Theoligonucleotide now may be used to conjugate with an activated proteincontaining a sulfhydryl-reactive group.

[1033] SPDP Modification of Amines on Superantigen Nucleotides

[1034] SAg DNA that has been modified with an amine-terminal spacer armmay be thiolated to contain a sulfhydryl residue. Theoretically, anyamine-reactive thiolation reagent may be used to convert an amino groupon a SAg DNA molecule into a thiol. A preferred reagent both forcross-linking and for thiolation reactions is the heterobifunctionalreagent SPDP. The NHS ester end of SPDP reacts with primary amine groupsto produce stable amide bonds. The other end of the cross-linkercontains a thiol-reactive pyridyldisulfide group that also can bereduced with DTT to create a free sulfhydryl. The reaction of a5′-diamine-modified SAg DNA oligonucleotide with SPDP proceeds undermildly alkaline conditions (optimal pH 7-9) yields thepyridyldisulfide-activated intermediate. This derivative can be used tocouple directly with sulfhydryl-containing compounds, or it may beconverted into a free sulfhydryl for coupling to thiol-reactivecompounds. In an alternative approach, 2,2′-dipyridyldisulfide is usedto create reactive pyridyldisulfide groups on a reduced5′-cystamine-labeled SAg oligonucleotide. This derivative then can beused to couple with sulfhydryl-containing molecules, forming a disulfidebond. Reduction of the pyridyldisulfide end after SPDP modificationreleases the pyridine-2-thione leaving group and generates a terminal-SHgroup.

[1035] Protocol

[1036] 1. Dissolve the amine-modified SAg oligonucleotide to bethiolated in 250 ml of 50 mM sodium phosphate, pH 7.5.

[1037] 2. Dissolve SPDP at a concentration of 6.2 mg/ml in DMSO to makea 20 mM stock solution. Alternatively, LC-SPDP may be used and dissolvedat a concentration of 8.5 mg/ml in DMSO (also makes a 20 mM solution).If the water-soluble Sulfo-LC-SPDP is used, a stock solution in watermay be prepared just prior to addition of an aliquot to the thiolationreaction. In this case, prepare a 10 mM solution of Sulfo-LC-SPDP bydissolving 5.2 mg/ml in water. Since an aqueous solution of thecross-linker will degrade by hydrolysis of the sulfo-NHS ester, itshould be used quickly.

[1038] 3. Add 50 ml of the SPDP (or LC-SPDP) solution to the SAgoligonucleotide solution. Add 100 ml of the Sulfo-LC-SPDP solution, ifthe water-soluble cross-linker is used. Mix.

[1039] 4. Allow to react for 1 h at room temperature.

[1040] 5. Remove excess reagents from the modified SAg oligonucleotideby gel filtration. The modified oligonucleotide now may be used toconjugate with a sulfhydryl-containing molecule, or it may be reduced tocreate a thiol for conjugation with sulfhydryl-reactive molecules.

[1041] 6. To release the pyridine-2-thione leaving group and form thefree sulfhydryl, add 20 ml of 1M DTT and incubate at room temperaturefor 15 mm. If present in sufficient quantity, the release ofpyridine-2-thione is followed by its characteristic absorbance at 343 nm(e=8.08×10³ M⁻¹ cm⁻¹). For many oligonucleotide modificationapplications, however, the leaving group will be present in too low aconcentration to be detectable.

[1042] 7. Purify the thiolated oligonucleotide from excess DTT bydialysis or gel filtration using 50 mM sodium phosphate, 1 mM EDTA, pH7.2. The modified oligonucleotide should be used immediately in aconjugation reaction to prevent sulfhydryl oxidation and formation ofdisulfide cross-links.

[1043] N-succinimidyl S-actylthioacetate (SATA ) Modification of Amineson Superantigen DNA Nucleotides

[1044] SAg oligonucleotides containing amine groups introduced byenzymatic or chemical means may be modified with SATA to produceprotected sulfhydryl derivatives. The NHS (N-hydroxylsuccinimide) esterend of SATA reacts with a primary amine to form a stable amide bond.After modification, the acetyl protecting group can be removed as neededby treatment with hydroxylamine under mildly alkaline conditions. Theresult is terminal sulfhydryl groups that can be used for subsequentlabeling with thiol-reactive probes or activated-protein derivatives.

[1045] Protocol

[1046] 1. Dissolve the amine-modified SAg oligonucleotide to bethiolated in 250 ml of 50 mM sodium phosphate, pH 8.

[1047] 2. Dissolve SATA in DMF at a concentration of 8 mg/ml.

[1048] 3. Add 250 ml of the SATA solution to the oligo solution. Mix.

[1049] 4. React for 3 h at 37° C.

[1050] 5. Remove excess reagents by gel filtration.

[1051] 6. To deprotect the thioacetyl group, add 100 ml of 50 mMhydroxylamine hydrochloride, 2.5 mM EDTA, pH 7.5, and react for 2 h.

[1052] 7. The sulfhydryl-containing oligonucleotide may be usedimmediately to conjugate with a sulfhydryl-reactive label, or it can bepurified from excess hydroxylamine by gel filtration.

[1053] Conjugation of a Polypeptide to SAg DNA

[1054] As indicated, the DNA molecule must be modified to contain one ormore suitable reactive groups, such as nucleophiles like amines orsulfhydryls. The modifications that employ enzymatic or chemical methodscan result in random incorporation of modification sites or can bedirected exclusively to one end of the DNA molecule, e.g., 5′ phosphatecoupling.

[1055] Some of the more common procedures for preparing DNA-polypeptideconjugates are given below.

[1056] Polypeptide (e.g., VT) Conjugation to Cystamine-Modified SAg DNAusing Amine- and Sulfhydryl-Reactive Heterobifunctional Cross-linkers

[1057] Cystamine groups are added to the 5′ phosphate of SAg DNA asdescribed above. Once a sulfhydryl-modified DNA has been prepared, thefollowing protocol may be used. The protein is activated with SPDP.Reacting the SAgic DNA probe in excess allows easy separation ofuncoupled SAg oligonucleotide from conjugated molecules.

[1058] Protocol

[1059] 1. Dissolve a 5′-sulfhydryl-modified SAg oligonucleotide in wateror 10 mM EDTA at a concentration of 0.05-25 mg/ml. Calculate the totalnanomoles of oligonucleotide present based on its molecular weight.

[1060] 2. Add 0. 15M NaCl, 10 mM EDTA, pH 7.2. Add the oligonucleotidesolution to the activated protein in a 10-fold molar excess.

[1061] 3. React at room temperature for 30 mm with gentle mixing.

[1062] 4. The protein-DNA conjugate is purified away from excess SAgoligonucleotide by dialysis or gel filtration, or through the use ofcentrifugal concentrators. Centricon-30 concentrators (Amicon) that havea molecular weight cutoff of 30,000 are also used to remove unreactedoligonucleotides. Since the polypeptide molecular weight isapproximately 140,000 and the conjugate is even higher, a relativelysmall DNA oligomer will pass through the membranes of these units whilethe conjugate will not. To purify the prepared conjugate usingCentricon-30s, add 2 ml of the phosphate buffer from step 2 to oneconcentrator unit, then add the reaction mixture to the buffer and mix.Centrifuge at 1000 g for 15 mm or until the retentate volume is about 50ml. Add another 2 ml of buffer and centrifuge again until the retentateis 50 ml. Invert the Centricon-30 unit and centrifuge to collect theretentate in the collection tube provided by the manufacturer.

[1063] Administration of Peptide-DNA (pDNA), Naked DNA, or Protein orPeptide Conjugates

[1064] Naked DNA, pDNA, nucleic acid-peptide or -polypeptide conjugatesor genetic fusion products are administered parenterally (for example,iv, ip, im, subcutaneously, intrathecally, intratumoral, rectally,transcutaneously) or orally. Administration may also be by a gene gunusing a 1 ml syringe and a 28 gauge needle. The nucleic acid isadministered intradermally or intramuscularly in a total volume of 100ml. A Tyne applicator is used to deliver doses of 1-1000 mg of DNA at 3×weekly intervals. SAg-encoding nucleic acid is injected directly intothe tumor. The nucleic acid either contains or does not containimmunostimulatory sequences that induce activation of T cells and skewthe response toward production of TH1 cytokines. For example, if nucleicacids encoding a tumor associated antigen are used then the nucleicacids are engineered to incorporate ISS sequences in order to fullyactivate a TH1 response. Likewise, if nucleic acid encoding a tumorassociated antigen is cotransfected with nucleic acid encoding a SAg,then one of the nucleic acid constructs is engineered to contain an ISS.

[1065] Viral DNA, nucleic acid expression cassettes or plasmids orbacteriophages encoding the constructs given in Table II may be used forin vivo immunization in place of naked DNA. Viruses may also acquire theαGal epitope after transfection into tumor cells which contain theα-galactosyltransferase enzyme either naturally or via transfection. Thevirus must possess the intact N-acetyllactosamine substrate for thegalactosyl-transferase in order to express the αGal. The virusesshedding from these cells will express the αGal epitope. The virus alsocontains peptide sequences for SAg and tumor associated antigen acquiredfrom the tumor cells which were previously transfected with nucleicacids encoding SAg and tumor antigen. The shed virus may also expressstaphylococcal or streptococcal hyaluronidase and capsularpolysaccharide sequences obtained from host tumor cell or accessorycells previously transfected with nucleic acids encoding these genes.The shed virus expressing αGal, SAg, hyaluronidase and capsularpolysaccharide is capable of initiating a potent tumoricidal responsewhen administered to hosts with established tumors or when used as atumor vaccine against potential tumors.

[1066] These constructs are also used as vaccines. Further, the nucleicacid construct is pre-processed ex vivo in muscle cells before selectivedelivery into host tumor tissue. Cationic liposomes or other liposomesor drug carriers well known in the art are used as vehicles to deliverthe nucleic acids in vivo.

[1067] The transfection process is also carried out ex vivo. Nucleicacids encoding SAgs together with the nucleic acid constructs given inTable II are transfected into tumor cells of all types and antigenpresenting cells such as MHC class I and class II as well as APCsexpressing CD1 and mannose receptors. These include but are not limitedto DCs, immunocytes, monocytes, macrophages, and fibroblasts. SAg istransfected alone or together with one or more of the above constructsgiven in Table II. The transfected cell expresses/secretespreferentially a SAg plus an immunogenic oncogene product,anti-angiogenesis factor, glycosylceramide, LPS or Gal. Thetransfectants present their gene products on cell surface receptors suchas conventional MHC molecules for SAgs or in the case of theglycosylceramides or LPS on a CD-1 or mannose receptor. (APC).Glycosylated SAgs show preference for presentation on mannose receptors.

EXAMPLE 4 SAgs, Tumor Antigens, Glycosylceramides, LPS's, Binary andTernary Complexes Applied to MHC Class I, Class II, CD1 or MannoseReceptors

[1068] The above molecules and all of the conjugates given in section 55are applied to antigen presenting receptors as given below. CD1represents a family of non-polymorphic antigen presenting moleculesunlinked to the MHC molecules expressed by most professional APCs. TheNKT cells that recognize CD1 presented antigens express NKR-P1, Ly49receptors, an invariant chain and a V8.2 variable region. With respectto these receptors, they share identity and their natural ligands withNK cells. Specifically, CD1 binds peptides with extended NH2 and COOHtermini flanking the core binding motif Long peptides (greater than 8 to10 amino acids) with amino acid residues at their hydrophobic bindingsites and greatly restricted anchors are preferred. This recognition ofCD1-presented antigens depends on the type and distribution of sugarresidues. Mycobacterial cell wall antigens namely mycolic acids andlipoarabinomannan also bind to CD1. Recently several glycosylceramides,in particular, monogalactosyl ceramides GalCer) were shown to bind toCD1 and to activate NKT cells Specifically, CD1 molecules are capable ofpresenting mannosides with 1,2 linkages and a phosphatidylinositol unit.CD1 bound antigens are recognized by NKT cells (/TCR positive; CD4 andCD8 negative). For instance, NKT cells are activated by alipoarabimannan (LAM) presented on CD1 receptors and become cytolyticwhile producing abundant INF.

[1069] In the present invention, a SAg bound to a monogalactosylceramidesuch as GalCer is loaded onto CD1 or MHC class I or II receptorsexpressed by APCs. The CD1 or MHC receptors are in soluble orimmobilized form produced by methods well described in the art.According to this invention, CD1 receptors present SAg polypeptidescomplexed with GalCer lipids or oligosaccharides to T cell and/or NKTcell population which recognize the conjugates and commencedifferentiation to tumor specific effector cells. These ligands are beloaded on the CD1 receptor sequentially, simultaneously or as apreformed conjugates. Alternatively, they are positioned on the CD1receptor after internal processing of their nucleic acid counterparts inthe antigen presenting cells. These cells are then harvested and usedfor adoptive immunotherapy (Examples 7, 15, 16. 18-23).

[1070] These complexes are also useful in vivo as a preventative ortherapeutic antitumor vaccine (Example 14, 15, 16, 18-23).

[1071] SAgs and tumor associated antigen (TAA) are loaded sequentiallyon to class II receptors of antigen presenting cells. Alternatively,preformed complexes of tumor associated antigen and SAg are loaded ontoMHC class II receptors. The SAg may be in the native or glycosylatedform. The tumor associated antigen is also fused genetically to the bchain of the MHC class II receptor. A SAg is added once the TAA isexpressed bound to the MHC class II. The sequence may also be reversedso that a SAg is genetically processed and bound to the b chain afterwhich the TAA is added. Consensus or repeating nucleic acid sequencesshared by a tumor associated antigen and a SAg are cloned into a singlesequence and transfected into APCs which display the consensus peptidein the context of the class II receptor. Methodology for production ofthe fusion genes is well described in the art. (See Ausubel. F M et al.,supra; Sambrook, J et al., supra) T cells or NKT cells are activatedafter exposure to SAg and TAA producing an expanded tumor specific Tcell effector population which is useful in adoptive immunotherapy ofcancer (Examples 7, 15. 16, 18-23).

[1072] Antigen presenting cells in this system are chosen from a groupconsisting of DCs, fibroblasts, macrophages, and lymphocytes, but otherprofessional APCs or any other cell transfectants, phage displays orliposomes expressing the class I or class II receptors are also used.Alternatively, a tumor associated antigen is bound to an APC that ispharmacologically or genetically inhibited from antigen processing. SAgis added and the complex of SAg and protein bound to class II is thenpresented to a T cell population to produce a tumor specific effectorcell population for use in adoptive immunotherapy of cancer as inExample 15, 16, 18-23). These complexes are also useful in vivo as apreventative or therapeutic antitumor vaccine (Example 14, 15, 16,18-23).

[1073] Soluble SAg MHC class II proteins with covalently bound singlepeptides are produced using a baculovirus system to express in insectcells two murine class II molecules with peptides attached by a linkerto the N terminus of their β-chains (Kozono H. et al., Nature 369:151-154 (1994)). The resulting peptide is engaged by the peptide bindinggroove of the secreted MHC molecule and this complex is recognized by Tcells bearing receptors specific for the combination. In this method,the approximately 100 bp fragment encoding the SAg and a flexible linkerwith an embedded thrombin cleavage site is introduced in frame by thePCR just after the third codon of the b1 domain. This assures arecognizable leader peptide cleavage site and flexible link between theC-terminus of the foreign peptide bound in the cleft of the MHC moleculeand the N terminus of the b1 domain of SAg amino acids. Solublecomplexes consisting of receptors and various SAg are prepared in thisway and are used to activate T cells for use in adoptive immunotherapy.Similarly, preparations consisting of MHC class I receptors, CD1 ormannose receptors complexed with SAgs, glycosylceramides or LPS's areproduced which are useful in activating T cells or NKT cells foradoptive immunotherapy of cancer in protocols given in Examples 7, 15,16, 18-23). These complexes are also useful in vivo as a preventative ortherapeutic antitumor vaccine (Example 14, 15, 16, 18-23).

[1074] To produce complexes composed of SAgs with class I or II MHC orsoluble DR a or b (lacking the transmembrane domain) and TCRheterodimer, a soluble human TCR heterodimer which has specificity forvarious tumor associated antigens bound to the human class I or II MHCmolecules or human soluble CD1 molecules is used. A typical system forpreparing ternary SAg-tumor peptide-MHC or ternary CD1-glycosylceramide(preferably GalCer)-SAg complexes capable of triggering T cells or NKTcells is as follows. CD1, DR-1 or HLA-A2 restricted tumor antigenspecific T cell or NKT cell clones are used although primaryunsensitized T or NKT cells may be used as well. The DR-1 and HLA-A2homozygous Epstein-Barr virus-transformed B cell line LG-2 or DCsexpressing CD1 receptors are used as APCs either live or fixed in 0.5%paraformaldehyde for 20 minutes. LG-2 and DCs (2.67×10⁵ per ml) in RPMI1640 with 1% fetal bovine serum are pulsed with tumor antigen andglycosylceramide respectively for 2 hours at 37° C. and then washed inRPMI 1640/1% fetal bovine serum to remove unbound antigen. SAg is addedfor 2 hours at 37° C. Pulsed APCs (4×10⁴ per well) are co cultured withresting T cells or NKT cells (2×10⁴ per well) in round-bottom microtiterplates in RPMI 1640/5% human serum. Twenty four hours later, the cellsare harvested. The APCs are separated and the T cells or NKT cells maybe optionally expanded further with IL.-2 Optionally, complexescomprising soluble recombinant DRα or β chain with bound superantigenare presented to the T cell or NKT cells which are then expanded withIL-2. These cells are then harvested and used for adoptive immunotherapy(Examples 7, 15, 16. 18-23). The APC containing the complexes are alsouseful in vivo as a preventative or therapeutic antitumor vaccine(Example 14, 15, 16, 18-23).

[1075] Also useful for tumor therapy are the complexes LIP⁺ GPI-SAg(from Section 38), either free or in the form of vesicles or exosomescomprising SAg-GalCer complexes or SAg-tumor peptide (including but notlimited to normal mutated structures). The ternary complexes ofSAg-GalCer-heat shock protein and tumor peptide-heat shock protein arealso useful, These complexes may be in or soluble or immobilized form,attached to a CD1 or MHC or as part of a vesicle or exosome. thecomplexes are also useful in vivo as a preventative or therapeuticantitumor vaccine (Example 14, 15, 16, 18-23).

[1076] The tumor associated antigen or SAg-tumor associated antigencomplex is conjugated to oxidized mannan (polymannose) by methodsdescribed by Apostolopoulos, V et al., Proc. Natl. Acad. Sci. USA 92:10128-10132 (1995) which is then loaded onto mannose receptors ofantigen presenting cells for stimulation of a T cell anti-tumorresponse. Alternatively, the SAg (optionally conjugated to tumorpeptides)-mannan conjugate is administered to tumor bearing hosts bymethods in Example 15, 16, 18-23).

[1077] The SAg alone or conjugated to a tumor associated antigen isrecognized by the mannose receptor on macrophages. This requires aglycosylated SAg which is recognized by the mannose receptor onmacrophages. A native or glycosylated tumor associated antigen-SAgconjugate or a consensus peptide of both polypeptides is presented tomannose receptors expressed on antigen presenting cells which areexposed to a T cell or NKT cell population to produce a tumor specificeffector cells by methods in Example 15, 16, 18-23). These complexes arealso useful in vivo as a preventative or therapeutic antitumor vaccine(Example 14, 15, 16, 18-23). They are also used ex vivo to produce apopulation of tumor specific effector T or NKT cells for the adoptiveimmunotherapy cancer by methods and protocols given in Examples 7, 15,16, 18-23 and 36.

[1078] The mannose receptor delivers the complex to the late endosomaland lysosomal vesicles and the MHC class II loading compartment wherethe antigen is loaded onto CD1b molecules. The C1b molecule isendocytosed at the plasma membrane in coated pits and vesiclestructures, transits to early endosomes and is then delivered to the MHCclass II antigen loading compartment. The endosomal localization motifon the tail of the CD1b molecule is essential for antigen trafficking ofCD1b through the lysosomal compartment required for loading of antigeninto CD1b and its ultimate transport to the membrane. The antigenbinding groove of CD1 is deeper and narrower than the MHC class Imolecule groove containing a hydrophobic binding site which accommodatesthe lipid portion of the molecules such as lipoarabinomannan or GalCerand the SAg-LPS constructs given herein. APCs expressing the aboveconstructs are exposed to NKT cell populations which recognize theantigens in the context of the CD1 receptor. If carried out ex vivo thisresults in the formation of tumor specific effector NKT cells which areused for adoptive immunotherapy by protocols given in Examples 7, 15,16, 18-23).

EXAMPLE 5 SAg Conjugation to Glycosylceramides, Gangliosides, LPS's,Glycans, Peptidoglycans Phytosphingolipids, Lipoproteins, oxyLDL andLipoarabinomannans

[1079] All of the SAg-lipid conjugates given above and in section 55 areprepared as follows. Selection of the SAg peptide to be used forcoupling is governed by several criteria. In practice, a 10-15 residuepeptide is selected. For SAgs, the sites chosen for coupling are thosepresumed not to be vitally involved in T cell binding and activation. Inmost SAgs, these sites are broadly distributed throughout the molecule.They are available at flexible regions of the protein and on reverseturns or loop structures. C termini are more mobile than the rest of themolecule and frequently exposed on the protein surface. This region isaccessible to be coupled to another ligand especially usingm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) via a Cys residuethat has been added to the N terminus of the peptide. By coupling thepeptide via its N-terminal end, the peptide is exposed in a fashionsimilar to that found in the native antigen.

[1080] Additional criteria for selection of the coupling site such asexposed hydrophilic regions, secondary structure, hydropathicityprofiles, and probability of helix formation may not be useful. However,care is take not to disrupt predicted polysaccharide attachment sites,most notably the sequence Asn-X-Ser or Asn-X-Thr, which predicts thepresence of Asn-linked polysaccharide moieties. In addition to locationof transmembrane regions, Asn-linked glycosylation sites and sites ofsignal sequence cleavage are all important. After due consideration, theC using 7-15 residues terminus is preferred and is modified toaccommodate MBS. This procedure requires a free sulfhydryl group on thesynthetic peptide and free amino groups on the ligand. Therefore, to usethis method, it is necessary to add a Cys residue to the C or N terminusof the peptide.

[1081] Biochemical Conjugation Methods: SAgs are conjugated topolysaccharide containing structures using several methods welldescribed in the art (Hermanson, G T, Bioconjugate Techniques, AcademicPress, San Diego, Calif. 1996). Two methods are given here one utilizingthe isolated complex carbohydrate obtained from the purified gangliosidewhich is then chemically conjugated to SAg and in another method whereinthe ganglioside and SAg are both incorporated into a liposomal membrane.Either method is used to produce complexes which are included within thescope of this invention. However they are by no means exhaustive of allthe techniques which could be employed to conjugate human tumor antigensto SAg molecules. Other conjugation strategies may be utilized toproduce an immunologically active complex as described by thisinvention. (See Offord, R E., in Protein Engineering, A R Rees, ed.,Oxford, 1992)

[1082] Direct Conjugation of Ganglioside, LPS or Peptidoglycan to SAgMolecules

[1083] 1. Ganglioside or LPS antigens are purified and are thendissolved in aqueous solution at pH 6.0 at a concentration of 1.0 mM/ml

[1084] 2. Endoglycoceramidase from Rhodococcus (Genzyme) is added to theganglioside solution to a level of 5 milliunits. The solution isincubated overnight at 37° C. with gentle agitation. Theendoglycoceramidase specifically cleaves at the ceramide-polysaccharidebond liberating ceramide and clipping off the complex carbohydratemaking up the ganglioside

[1085] 3. The polysaccharide is isolated by HPLC size exclusionchromatography or by ultrafiltration

[1086] 4. SAg is dissolved in IM sodium phosphate, 0.15 M NaCl, pH 7.5,at a concentration of 1 mg/ml. The purified polysaccharide antigen isadded to this solution to a concentration of at least 1 mM/ml.

[1087] 5. In a fume hood, 20 microliters of 5M sodium cyanoborohydridesolution in 1M NaOH (Aldrich) is added to each ml of the SAg solution.

[1088] 6. The reaction is mixed gently and incubated at room temperaturefor 72 hours or 4° C. for 1 week. This reaction reductively aminates thereducing end of the polysaccharide (at the point it was cleaved by theendoglycoceramidase) to the amine groups on the SAg protein creatingstable conjugate coupled through a secondary amine linkage. The degreeof polysaccharide coupling can be controlled by limiting the time ofreaction.

[1089] 7. Remove unreacted carbohydrate and cyanoborohydride by gelfiltration on Sephadex G-25 or by dialysis.

[1090] In a second method, SAg-GalCer, SAg-GalCer-CD1,SAg-glycosphingolipid, or SAg-glycosphingolipid-CD1 complexes or SAgconjugates given in section 55 are produced which have the added benefitof presenting the glycosylceramide in a polyvalent array which isimportant for high affinity binding to complementary receptors. Theyretain nearly all of their original structure including most of theceramide moiety and the entire oligosaccharide chain. The principle ofpreparation derived from Mahoney, J A et al., Meth. Enzymol 242: 17-27(1994) is as follows. The fatty acid amide is hydrolyzed from the intactganglioside converting it to its lyso form which has a unique primaryamine at the 2-position of sphingosine. The lysoganglioside is treatedwith a bifunctional cross-linking reagent, succinimidyl4(N-maleimidomethyl)cyclohexane 1-carboxylate (SMCC), which forms anamide bond to the 2-position of sphingosine and results in asulfhydryl-reactive maleimidyl moiety attached through a linker arm, tothe original position of the fatty acid amide on the ceramide portion ofthe ganglioside. The SAg protein is treated with a reagent,N-succinimidyl S-acetylthioacetate (SATA), which converts the lysinee-amino groups to acetylated sulfhydryls. Subsequent treatment withhydroxylamine reveals the desired free sulfhydryls. Treatment ofsulfhydryl-derivatized SAg with maleimidyl derivatized gangliosideresults in a stable thioester linkage between the ganglioside and theprotein. The final product is chromatographically purified andcharacterized by protein and carbohydrate analysis. The SAg-GalCer orSAg-glycosphingolipid complex is then loaded onto a soluble CD1receptor.

[1091] LPS's and peptidoglycans are conjugated to SAg by methods welldescribed in the art. The most convenient and preferred method to targetspecifically the polysaccharides on the protein is through mild sodiumperiodate oxidation. Periodate cleaves adjacent hydroxyl groups in sugarresidues to create highly reactive aldehyde functional groups. Thegenerated aldehydes are used to in coupling reactions with amine orhydrazide containing molecules to form covalent linkages. Amines reactwith formyl groups under reductive amination conditions using a suitablereducing agent such as sodium cyanoborohydride. The result of thereaction is a stable secondary amine linkage. Hydrazides spontaneouslyreact with aldehydes to form hydrazone linkages, although the additionof a reducing agent greatly increases the efficiency of the reaction andthe stability of the bond. (See Hermanson, G T. Bioconjugate Techniques,Academic Press, San Diego Calif. 1996).

[1092] Production of Liposomes Displaying Glycolipid or Apolipoproteinor oxyLDL-SAg Complexes

[1093] Liposomes composed of the highly immunogenic constructs describedherein are prepared. They may include lipoproteins such as SAgs coupledto Gal, GalCer, SAg-glycosphingolipid, SAg-glycosylceramides,SAg-phytosphingolipids, SAg-mycosphingolipid and SAg-lipid conjugatesgiven in section 55. Liposomes comprising SAgs conjugated toapolipoproteins or oxyLDL receptors are useful for targeting endothelialor macrophage oxyLDL receptors in tumor microvasculature. Other SAgconjugates e.g., SAg-glycosphingolipid, SAg-glycosylceramides,SAg-phytosphingolipids, SAg-mycosphingolipid are useful in immunizing Tcells, NK cells and NKT cells. Cationic liposomes are also useful as ameans of transferring the nucleic acid constructs of this invention totumor tissue. GalCer (a monogalactosylceramide) comprises the majorportion of the liposome. The most effective lengths of fatty acyl chainand sphingosine (or ceramide) base are C26 and C18 respectively and aphytosphingosine backbone. Sphingolipids lend structural advantages tothe integrity of liposomal membranes and have prolonged duration invivo. The Gal carbohydrate epitope is linked to liposomes via theamphipathic properties of the surface sphingolipids. The Gal isconverted to a glycolipid with a sphingosine backbone possessing ahydrophobic fatty acid tail that embeds them into membrane bilayers. Thehydrophilic carbohydrate ends of these amphipathic molecules caninteract with molecules dissolved in the surrounding environment.Sphingosine glycolipids consisting of lactosylceramide,GalGal(1-3)Gal(1-4)GlcNAc-R) or glycosphingolipids with terminalGal(α1-4)Gal are prepared in a manner similar to that of sphingolipids.

[1094] All methods of preparation of liposomes have several steps incommon: (1) dissolution of the lipid mixture in an organic solvent, (2)dispersion in an aqueous phase, and (3) fractionation to isolate thecorrect liposomal population.

[1095] In the first stage, the desired mix of lipid components isdissolved in organic solvent (usually chloroform:methanol (2:1 byvolume) to create a homogenous mixture. This mixture includes anyphospholipid derivatized to contain reactive groups as well as otherlipids used to form and stabilize the bulk of the liposomal structure.The correct ratio of lipid constituents to form stable liposomes isimportant A reliable liposomal composition for encapsulating aqueoussubstances contains molar ratios of lecithin:cholesterol:negativelycharged phospholipid (e.g., phosphatidyl glycerol) of 0.9:1:0.1.Apolipoproteins (e.g., LP(a)) or oxyLDL (e.g., 7β-hydroperoxycholesterolor 7β-hydroperoxy-choles-5-en-3β-ol) can substitute for cholesterol inthe preparation of the liposomes. In general, to maintain membranestability, the PE derivative should not exceed a concentration ratio ofabout 1-10 mol PE per 100 mol of total lipid. Once the desired mixtureof lipid components is dissolved and homogenized in organic solvent,several techniques are used to disperse the liposomes in aqueoussolution. These methods are broadly classified as (1) mechanicaldispersion, (2) detergent-assisted solubilization, and (3)solvent-mediated dispersion. With mechanical dispersion to formvesicles, the lipid solution is dried to remove all traces of organicsolvent prior to dispersion in aqueous media. The dispersion process iskey to producing liposomal membranes of the correct morphology. Methodsutilized include simple shaking, high pressure emulsification,sonication, extrusion through small-pores membranes and variousfreeze-thaw techniques. Detergent-assisted solubilization is also usedto bring the lipid more effectively into the aqueous phase fordispersion. Triton X, alkyl glycosides or bile salts such as sodiumdeoxycholate are employed. Other modalities or dispersion include thesteps of dissolving phospholipids and other lipid to be part of theliposomal membrane in ethanol. This ethanolic solution is then rapidlyinjected into an aqueous solution of 0.16 M KCl using a Hamilton syringeresulting in a maximum concentration of no more than 7.5% ethanol. Usingthis method, single bilayer liposomes of about 25-nm diameter areproduced. To remove the excess aqueous components that are notencapsulated during the vesicle formation, gel filtration using SephadexG-50 or dialysis is employed. To fractionate the liposome populationaccording to size, gel filtration is carried out using a column ofSepharose 2B or 4B

[1096] SAgs are conjugated to the GalCer or glycosphingolipids withterminal Gal(a1-4)Gal, apolipoproteins, LDL or oxyLDL or LDL receptorsbefore incorporation into the liposomal membrane or they may beincorporated into the membrane during the preparation of the liposomalmembrane. Likewise, the SAg is conjugated to GalCer orglycosphingolipids with terminal Gal(a1-4)Gal at the glycolipid's polarhead region by methods well known in the art including usingheterobifunctional crosslinkers or periodate oxidation techniques.Alternatively, after the GalCer or glycosphingolipids with terminalGal(a1-4)Gal is incorporated into the membrane, the liposomes arederivatized for further binding to the SAg proteins using the sodiumperiodate which oxidizes the ceramide's free hydroxyl to an aldehydewhich is further modified by reductive amination. Using thephosphatidylethanolamine of the lipid in the liposome, SAgs are coupledto the liposome using various bifunctional agents includingcarbodiimide, glutaraldehyde, dimethyl suberimidate, periodate oxidationfollowed by reductive amination, N-succinimidyl3-(2-pyridyldithio)propionate (SPDP),succinmidyl-4-(p-maleimidophenyl)butyrate (SMPB), iodoacetate,succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC).

[1097] Two general methods are used to prepare immunogenic (i)SAg-GalCer, (ii) GalCerGal, (iii) GalCerGal-SAg and (iv)SAg-glycosphingolipid complexes: The molecules (1) are dissolved insolution and encapsulated within the vesicle construction, or (2)covalently coupled to phospholipid constituents in the lipids usingstandard cross-linking chemical reactions. Covalent coupling of SAg toliposomes is done through the head groups using various phospholipidderivatives and cross-linking chemical reactions. These are done via thePE molecules. Simple encapsulation is also a viable technique asdescribed in Hermanson (supra).

[1098] A sample method using periodate oxidation and reductive aminationis given below.

[1099] 1. A 5 mg/ml liposome suspension is prepared in 20 mM sodiumphosphate 0.15 M NaCl, pH 7.4. containing, on a molar ratio basis asmixture of phosphatidyl choline: cholesterol:phosphatidyl glycerol of8:10:1. Other liposome compositions may be used, for example methodswithout cholesterol, as long as a periodate-oxidizable componentcontaining vicinal hydroxyls (e.g., phosphatidyl glycerol) is present.Any method of liposome formation may be used that is common to thoseskilled in the art including mechanical dispersion.

[1100] 2. Sodium periodate is dissolved to a concentration of 0.6 M byadding 128 mg/ml of water. 200 ml of this stock periodate solution isadded to each mol of the liposome suspension with stirring.

[1101] 3. React for 30 min. at room temperature in the dark.

[1102] 4. The oxidized liposomes are dialyzed against 20 mM sodiumborate, 0.15 M NaCl, pH 8.4, to remove unreacted periodate. This bufferis ideal for the subsequent coupling reaction. Chromatographicpurification using a column of Sephadex G50 is also done. Theperiodate-oxidized liposomes are used immediately to couple with SAgmolecules or they may be stored in a lyophilized state in the presenceof sorbitol for later use.

[1103] 5. SAg is added to the periodate oxidized liposome solution toobtain a 1 mg/ml concentration.

[1104] 6. In a fume hood, add 20 ml of 5 M sodium cyanoborohydridesolution in 1 M NaOH (Aldrich) to each ml of the SAg solution.

[1105] 7. The reaction is mixed gently and incubated at room temperaturefor 6 hours.

[1106] 8. Excess SAg and cyanoborohydride are removed by size exclusionchromatography on a column of Sephadex G-50 or by dialysis using amembrane with a molecular weight cutoff of 100,000 daltons.

[1107] 9. Ganglioside antigens isolated by the method describedpreviously are incorporated into SAg-containing liposomes by detergentdialysis. An amount of ganglioside is added representing twice theamount of phosphatidyl glycerol (on a molar basis) originally added toform the liposome (prior to periodate oxidation). To this solution,concentrated sodium deoxycholate is added to obtain a finalconcentration of 0.7% (w/w) and mixed thoroughly using a Vortex mixer.Finally, the liposome suspension is dialyzed against PBS, pH 7.5. Asample of the encapsulation technique is given in Hermanson, supra.

[1108] An additional method for preparation of liposomes containingGalCer or glycosphingolipids with terminal Gal(a1-4)Gal is as follows:The donor liposomes consist of liver phosphatidylcholine, dicetylphosphate, cholesterol, 3-(Man1-3Man-sn-1,2diacylglycerol) andgalactosylceramide. These are mixed in various percentages to permitoptimal expression of the galactosylceramide. Constituent lipids inchloroform-methanol are mixed and dried under a stream of nitrogen.Buffer consisting of 0.15M NaCl, 10 MM sodium Phosphate, pH 7.4, 1 mMdithiothreitol, 0.02% NaN3 is added to the dried lipids at a volume of 1ml per 0.9 mmol of lipid phosphorus in the donor liposomes. After a30-min incubation at 25° C., the lipids are dispersed into the buffer bysonication with a Bransom sonifier for 30 min under nitrogen at 0° C.The liposome suspension is used the same day after centrifugation at1500 g for 30 min to remove any undispersed lipid and titanium fragmentsreleased from the sonication probe.

[1109] Liposomes used for transfer of nucleic acid constructs givenherein have unique structures as described below. A cationic liposomecomposed of dimyristyloxypropyl-3-dimethyl-hydroxyl ammonium (DMRIE)with DOPE has allowed up to 100 fold higher concentrations of lipid andDNA to be administered in vivo with minimal toxicity. Improvedtransfection techniques have been observed with the DMRIE/DOPE of two toseven fold. The prototype cationic lipid for gene transfer is DOTMA(N[1-(2,3-dioleyloxy)propyl]-N,N,N-tri-methylammonium chloride) which ismixed with a equimolar amount of DOPE (dioleoylphosphatidylethanolamine). The lipid DOTMA/DOPE comprise the cationicliposome known a Lipofectin. For human studies, two different cationicliposomes formulations are used. The first includesDC-cholesterol(3b[N-(N′N′-dimethylaminoethane)-carbamoyl] cholesterol)mixed with DOPE. DC-cholesterol/DOPE is low concentrations has proven toreduce toxicity to cells in vitro, is metabolized in vivo, and hasprovided successful gene transfer into malignant tumors in humans (SeeExample 17 for use in humans).

[1110] Genetic Fusion of SAgs to LPS's

[1111] N-linked glycosylation occurs exclusively in the ER, whereGlc3Man9GlcNAc2 is added to Asn residues present in the sequence Asn XSer/Thr (X, any residue except Pro). To produce a glycosylation site ona SAg capable of binding a LPS, recombinant vaccinia virus expressingSAg is produced with Gln149 or Asn149 directed to the ER by appendage ofNH2-terminal ER insertion, The SAg is directed to the secretory pathwayusing signal sequence from IFN. Recombinant vaccinia viruses(rVVs)expressing TAP and SAg nucleoprotein are used. The full length SAg genemodified by standard molecular genetic methods to encode glycosylationsites is inserted into the thymidine kinase locus of vaccinia viruses(VVs) by homologous recombination as described using the pSX11 plasmidto express foreign proteins under the control of the VV p7.5 early/latepromoter. SAg nucleoprotein is directed to the secretory pathway usingthe signal sequence from IFN-β. The SAg coding sequences of all of therVVs are verified by sequencing PCR-amplified copies of full-length NPgenes isolated from the rVV. The resulting SAg-LPS or SAg-lipoproteincomplexes are used to immunize a population of T or NKT effector cellsfor use in the adoptive immunotherapy of cancer (Examples 2, 5, 7 15,16, 18-23). They may be preloaded onto CD1 or MHC Class I or IIreceptors on APCs as described below in the course of ex vivoimmunization. These complexes may also be used in vivo as a preventativeor therapeutic antitumor vaccine as in Example 14, 15, 16, 18-23).

[1112] Preparation of Fusion Proteins

[1113] Preferred fusion proteins comprise SAgs linked to other proteinsor peptides such as VTs or their A and B subunits, IFNα receptors, CD19peptides or carbohydrate recognition units which are designed to targetthe SAg to glycosphingolipid receptors on tumor cells or α_(v)β₃ ligandArg-Gly-Asp or avβ5 ligand Asn-Gly-Arg in vivo or in vitro. These fusionproteins induce apoptosis of the tumor cells. The fusion proteins areproduced by conventional methods in a variety of cells using a varietyof vectors such as phage 1 regulatory sequences. Techniques are wellestablished for producing fusion proteins that include the lacZprotein(β-galactosidase), trpE protein, glutathione-S-transferase, andthioredoxin. Expression in E. coli is most conventional but baculoviralexpression systems are also useful. Fusion proteins are produced inbacteria by placing a strong, regulated promoter and an efficientribosome-binding site upstream of the cloned gene. Exemplified below isa procedure using a representative lacZ vector. However, it should berecognized that other vectors well known in the art would be useful.Plasmids encoding the above proteins are prepared as previouslydescribed.

[1114] Construction of Expression Plasmids and Detection of FusionProteins

[1115] 1. The appropriate pUR (or pEX or pMR100) vector is ligatedin-frame to cDNA fragments to be expressed as fusion partners using theabove plasmids to create an in-frame fusion. cDNA encoding theverotoxins may be obtained from Dr. G. Lingwood, University of Toronto;murine p31 Ii are from Dr. R. Germain, National Institutes of Health andJ. Miller, University of Chicago.

[1116] 2. Bacteria of the following strains are transformed: E. coli K1271/18 or JM1O3 with pUR vectors, M5219 with pEX vectors or LG9O forpMR100 vectors. The cells are plated on LB medium containing ampicillin(100 mg/ml) and incubated overnight at 37° C. (or 30° C. in the case ofthe pEX vector). MacConkey lactose indicator plates should be used forpMR100.

[1117] 3. Individual colonies are tested for the presence of the desiredinsert by plasmid minipreps. If most of the colonies can be assumed tocontain a cDNA (because directional cloning or a dephosphorylated vectorwas used in step 1), they can be screened for protein production inparallel (see step 4b). If not, clones that contain a cDNA, asdetermined by plasmid minipreps, can be screened for protein expressionlater. cDNA inserts into a pMR100 plasmid can be detected readily as redcolonies on the MacConkey lactose indicator plates.

[1118] 4. Colonies are screened as follows for expression of the fusionprotein.

[1119] a. Grow small cultures from 5-10 colonies in LB medium containingampicillin (100 mg/ml). Incubate overnight at 37° C. (or at 30° C. forpEX).

[1120] b. Inoculate 5 ml of LB medium containing ampicillin (100 mg/ml)with 50 ml of each overnight culture. Incubate for 2 hours at 37° C. (orat 30° C. for pEX) with aeration. Remove 1 ml of uninduced culture,place it in a microfuge tube, and process as described in steps d and e.If screening for protein production is being done in parallel, prepareplasmid minipreps from 1-ml aliquots of the overnight cultures.

[1121] c. Induce each culture as follows: For pUR or pMR100 vectors, addisopropylthio-β-D-galactoside (IPTG) to a final concentration of 1 nMand continue incubation at 37° C. with aeration. For pEX vectors,transfer the culture to 40° C. and continue incubating with aeration.

[1122] d. At various time points during the incubation (i.e., 1, 2, 3,and 4 hours), transfer 1 ml of each culture to a microfuge tube, andcentrifuge at 12,000 g for 1 minute at room temperature in a microfuge.Remove the supernatant by aspiration. The kinetics of induction varieswith different proteins, so it is necessary to determine the time atwhich the maximum amount of product is produced.

[1123] e. Resuspend each pellet in 100 ml of 1×SDS gel-loading buffer,heat to 100° C. for 3 minutes, and then centrifuge at 12,000 g for 1minute at room temperature. Load 15 ml of each suspension on a 6% SDSpolyacrylamide gel. Use suspensions of cells containing the vector aloneas a control. (For pEX and ORF vectors, also use β-galactosidase as acontrol.) The fusion protein should appear as a novel band migratingmore slowly than the intense β-galactosidase band in the control. It isnot uncommon for a protein the size of β-galactosidase to be presentalong with the fusion protein.

[1124] Composition of 1×SDS Gel-Loading Buffer

[1125] 50 mM Tris Cl (pH 6.8)

[1126] 100 mM dithiothreitol (DTT)

[1127] 2% SDS (electrophoresis grade)

[1128] 0.1% bromophenol blue

[1129] 10% glycerol

[1130] 1×SDS gel-loading buffer lacking dithiothreitol can be stored atroom temperature.

[1131] Dithiothreitol should then be added, just before the buffer isused, from a 1 M stock.

[1132] Loading of SAg-LPS, SAg-Lipoprotein, SAg-Lipid, SAg-Glycolipid orSAg-Phytolipid Conjugates onto CD1 or MHC Receptors

[1133] For loading of SAg LPS or SAg-lipoprotein SAg-lipid,SAg-glycolipid SAg-phytolipid conjugates or SAg-lipid complexes fromsection 55 onto CD1 receptors, recombinant soluble CD1-2M complexes inDrosophila melanogaster cells are used to screen a random peptide phagedisplay library(RPPDL). The absence of peptide-loading machinery in D.melanogaster cells results in the expression of class 1 molecules thatare properly folded and functionally competent but essentially devoid ofbound peptide. This approach has been shown to be useful in definingpeptide binding motifs for classical and nonclassical MHC Class I andClass II molecules. (Jackson et al., Proc. Natl. Acad. Sci.89:1217-1224, 1992; Hammer et al., J. Exp. Med 175,1007-1012, 1992;Hammer et al., Cell 74, 197-201, 1993). Each clone of SAg-lipoproteincontains a random 22-amino acid sequence at the mature NH2 terminus ofthe gene VIII (filamentous coat protein of the M13 bacteriophage).Recombinant soluble mCD1 is engineered with a C-terminal hemagglutinin(HA) tag, an epitope derived from the influenza HA protein. In this way,the mCD 1-phage complexes are identified with a HA tag specificantibody. For immunizing usage, isolated receptor or antigen presentingcells of various types which express CD1 or MHC class II moleculepretreated with formaldehyde may be used for loading the SAg-LPS orSAg-lipoprotein complexes. These APCs with bound complexes are then usedto immunize T cells or NKT cells for use in adoptive immunotherapy ofcancer (Examples 2, 7, 15, 16 18-23).

[1134] Incorporation of Exogenous Lipid e.g. Glycolipid,Phytosphingosine, Apolipoprotein or oxyLDL into Cells by Fusion withLiposomes

[1135] Liposomes. To prepare glycolipid, phytosphingosine,apolipoprotein, oxyLDL or receptor containing liposomes, 400 mg ofgalabiosylceramide (Gb2) globotriosylceramide (Gb3),globotetraosylceramide (Gb4), galactosylceramide (GalCer),glucosylceramide (GlcCer), phytoshpingosine, oxyLDL or apolipoproteinare dried with 200 mg of phosphatidylethanolamine (PE) and 200 mg ofphosphatidylserine (PS) under a stream of nitrogen gas. 400 ml ofsterile isotonic PBS, pH 7.4, is added to the lipid, and the mixture issonicated using a water bath sonicator for 30 minutes. Liposomepreparations are used immediately.

[1136] To incorporate exogenous glycolipid into cells, tumor cells inlate logarithmic growth phase, sickled erythrocytes or vesicles (1.6×10⁷cells) are washed twice with PBS to remove serum proteins and thensuspended in serum-free RPMI 1640 medium at 4×10⁶ cells/ml. The cellsare incubated in the presence of the liposomes (or PBS for controls)prepared as above with rotary shaking (100 rpm) at 37° C. for 1 hr.,washed twice (5 min. 800×g) with PBS. and incubated for 18-24 hr at 37°C. in the presence of medium supplemented with 10% fetal calf serumprior to use.

[1137] Membrane & Vesicle Transfers. Exogenous lipids e.g.,galactosylceramides from proximal tubular epithelial cells, MDCK caninerenal cells or phytosphinosine from amphibian cells are incorporatedinto dendritic cells or tumor cells or dendritic cell (accessorycell)/tumor cell fusions as follows:

[1138] Preparation of Plasma Membrane Plasma membrane fractions areprepared and analyzed as described (Monneron A et al., J Cell Biol. 77:211-231 (1978)). Only the lightest fractions (band 1 at the interfacebetween buffer and 22.5% sucrose and band 2 at the interface between22.5% and 35% sucrose), containing pure plasma membrane vesicles, arecollected and pooled. From 10¹⁰ cells, 0.8-1.0 mg of protein isrecovered in the combined fraction. The plasma membrane enzyme markers5′-nucleotidase (EC 3.1.3.5) and alkaline phosphatase (EC 3.1.3.1) areenriched 30-fold and 70-fold, respectively, over the homogenate; noactivity of the cytoplasmic enzyme lactate dehydrogenase are found. Notraces of RNA or DNA contamination are found. The membranes obtained areeither immediately frozen in aliquots at −70° C. or used at once.

[1139] Reconstituted Vesicles. Three types of vesicles are prepared asfollows: (i) reconstitution of solubilized Sendai virus envelopes (Vvesicles), (ii) reconstitution of solubilized plasma membranes (PMvesicles), and (iii) core constitution of solubilized Sendai virusenvelope and plasma membranes (VPM vesicles). Sendai virus (20 mg ofprotein), solubilized in 2 ml of resolution buffer containing 2% TritonX-100 for 1 hr at room temperature, is centrifuged (1 hr. 100,000×g) toyield a clear supernatant (1.5 mg of protein per ml) containing mainlythe two envelope glycoproteins, neuraminidase/hemagglutinin glycoprotein(NH) and fusion glycoprotein (F). For preparation of VPM vesicles,freshly solubilized plasma membranes (1 mg of protein per 0.3 ml ofresolution buffer containing 1% Triton X-100, 20 min, 4° C.) are mixedwith the solubilized viral envelopes (1 mg of protein per 0.7 ml ofresolution buffer) and dialyzed in Spectrapor membrane-2 against1000-fold excess of reconstitution buffer containing 1.5 g of wetBio-beads SM-2 per liter [for efficient removal of the detergent]. PMand V vesicles are prepared by the same procedure except that one of thecomponents was omitted and replaced by resolution buffer alone. Dialysisis continued for 96 hr at 4° C. until the Triton X-100 concentrationdropped below 0.02%. The dialyzed solutions are centrifuged at 100.000×gfor 3 hr at 4° C. to obtain reconstituted vesicles. The pellet issuspended in 0.3-0.5 ml of resolution buffer, divided into smallaliquots, and frozen at −70° C. Each aliquot is thawed only once forfusion experiments.

[1140] Fusion of Reconstituted Vesicles with Tumor Cells: Tumor cells(10⁶ cells) are incubated with 5-30 mg of protein of reconstitutedvesicles in 1 ml of fusion solution. After 60 mm at 4° C. withoccasional shaking, during which the Sendai virus bound to the cells,the cells are collected by centrifugation, resuspended in 1 ml of fusionsolution containing 5 mM Ca²⁺, and incubated at 37° C. for 30 mm for thefusion process. The cells are then washed with phosphate-buffered saline(pH 7.4) and suspended in RPMI-1640 medium containing 5% (vol/vol)heat-inactivated fetal calf serum.

[1141] Assessment of Incorporation: Tumor cells are analyzed for uptakeof the various glycolipids, apolipoproteins, oxyLDL, phytosphingosine,glycosylceramides by use of direct staining with specific antisera,binding by lectin receptors and they are tested in vitro for activationof T cells, NK cells, NKT cells using proliferative assays and inductionof cytokines IL-2, IL-4, IL-12 and IFN-g by methods well established inthe art.

EXAMPLE 6 Targeting SAg Nucleic Acids, Phase Display Systems andPolypeptides to Tumor Sites

[1142] Parenterally administered nucleic acid is targeted to aparticular cell population as follows. Nucleic acid is attached to adesialylated galactose moiety that targets asialo-orosomucoid receptorsin liver cells. Nucleic acid is attached to other ligands such astransferrin and TAP-1 as well as antibodies to surface structures suchas the Le^(v) receptor. These ligands and antibodies bind to surfacestructures and are internalized. Thus, the attached nucleic acid isdelivered to a cell of choice.

[1143] Sickled Erythrocytes as Gene Carriers

[1144] Erythrocytes from patients with sickle cell anemia contain a highpercentage of SS hemoglobin which under conditions of deoxygenationaggregate followed by the growth and alignment of fibers transformingthe cell into a classic sickle shape. Retardation of the transit time ofsickled erythrocytes results in vaso-occlusion. SS red blood cells havean adherent surface and attach more readily than normal cells tomonolayers of cultured tumor endothelial cells. Reticulocytes frompatients with SS disease have on their surface the integrin complex a4b1which binds to both fibronectin and VCAM-1, a molecule expressed on thesurface of tumor endothelial cells particularly after activation byinflammatory cytokines such as TNF, interleukins and lipid-mediatedagonists (prostacyclins). Activated tumor endothelial cells aretypically procoagulant. Similar molecules are upregulated on theneovasculature of tumors. In addition, upregulation of the adhesive andhemostatic properties of tumor endothelial cells are induced by viruses,such as herpes virus and Sendai virus. Sickled erythrocytes lackstructural malleability and aggregate in the small tortuousmicrovasculature and sinusoids of tumors. In addition, the relativehypoxemia of the interior of tumors induces aggregation of sicklederythrocytes in tumor microvasculature. Hence, sickled erythrocytes withtheir proclivity to aggregate and bind to the tumor endothelium areideal carriers of therapeutic genes to tumor cells.

[1145] Red blood cell mediated transfection is used to introduce variousnucleic acids into the sickled erythrocytes. The extremely plasticstructure of the erythrocyte and the ability to remove its cytoplasmiccontents and reseal the plasma membranes enable the entrapment ofdifferent macromolecules within the so-called hemoglobin free “ghost.”Combining these ghosts and a fusogen such as polyethylene glycol haspermitted the introduction of a variety of macromolecules into mammaliancells (Wiberg, F C et al., Nucleic Acid Res. 11: 7287-7289 (1983);Wiberg, F C et al., Mol. Cell. Biol. 6: 653-658 (1986); Wiberg, F C etal., Exp. Cell. Res. 173: 218-227 (1987). Both transient and stableexpression of introduced DNA are achieved by this method. Sickled cellscan also be transfected with a nucleic acid of choice e.g.,apolipoproteins, RGD in the nucleated prereticulocyte phase (e.g.proerythroblast or normoblast stage) by methods given in Example 1.Sickled erythrocytes transfected with nucleic acids encoding a SAgand/or carbohydrate modifying enzyme to induce expression of the α-Galepitope, apolipoproteins, RGD and/or any construct described herein.Nucleic acids encoding additional polypeptides alone or together withSAg as described in Tables I and II to including but not limited toangiostatin, apolipoproteins, RGD, streptococcal or staphylococcalhyaluronidase, chemokines, chemoattractants and Staphylococcal protein Aare transfected into and expressed by sickled erythrocytes. Thesesickled cell transfectants are administered parenterally and localize totumor neovascular endothelial sites where they induce a anti-tumorresponse. The methods of in vivo transfection of tumor cells are givenin the Examples 17. Protocols for use of these transfectants in theinduction of anti-tumor immune response are described in Examples 14,15, 16, 18-23, 31.

[1146] Vesicles from Sickled Erythrocytes

[1147] Vesicles from sickled erythrocytes are shed from the parentcells. The contain membrane phospholipids which are similar to theparent cells but are depleted of spectrin. They also demonstrate that ashortened Russell's viper venom clotting time by 55% to 70% of controlvalues and become more rigid under acid pH conditions. Rigid sickle cellvesicles induce hypercoagulability, are unable to pass through thesplenic circulation from which they are rapidly removed. Sicklederythrocytes are transfected in the nucleated prereticulocyte phase withsuperantigen and apolipoprotein nucleic acids as well as RGD nucleicacids. Nucleic acids encoding additional polypeptides alone or togetherwith SAg as described in Tables I and II are transfected into andexpressed by sickled erythrocytes. Any of the immature or mature sicklederythrocytes and their shed vesicles expressing the molecules given inTables I and II are capable of localizing to tumor microvascular siteswhere they bind to apolipoprotein receptors and induce an anti-tumoreffect. Because of their adhesive and hypercoagulable properties as wellas their rigid structure, these sickled cell vesicles expressingsuperantigen and apolipoproteins are especially useful for targeting thetumor microvascular endothelium and producing a prothrombotic,inflammatory anti tumor effect. Sickled erythrocytes and their vesiclesare capable of acquiring oxyLDL via fusion with oxyLDL containingliposomes as in Example 5. The resulting sickle cell or liposomeexpresses oxyLDL alone or together with SAg. Binding of oxyLDL to theSREC receptor on tumor microvascular endothelial cells induces apoptosisand simultaneous superantigen deposition produces a potent T cellanti-tumor effect.

[1148] Vesicles are prepared and isolated as follows: Blood is obtainedfrom patients with homozygous sickle cell anaemia. The PCV range is20-30%, reticulocyte range is 8-27%, fetal hemoglobin range is 25-13%and endogenous level of ISCs is 2-8%. Blood is collected in heparin andthe red cells are separated by centrifugation and washed three timeswith 09% saline. Cells are incubated at 37° C. and 10% PCV inKrebs-Ringer solutions in which the normal bicarbonate buffer isreplaced by 20 mM Hepes-NaOH buffer and which contains either 1 mM CaCl2or 1 mM EGTA. All solutions contain penicillin (200 u/ml) andstreptomycin sulphate (100 μg/ml). Control samples of normalerythrocytes are incubated in parallel with the sickle cells.Incubations of 10 ml aliquots are conducted in either 100% N₂ or in roomair for various periods in a shaking water bath (100 oscillations permm). N₂ overlaying is obtained by allowing specimens to equilibrate for45 mm in a sealed glove box (Gallenkamp) which was flushed with 100% N₂.Residual oxygen tension in the sealed box was less than 1 mmHg. Thepercentage of irreversibly sickled cells is determined by counting. 1000cells-after oxygenation in room air for 30 mm and fixation in bufferedsaline (130 mM Cl, 20 mM sodium phosphate, pH 74) containing 2%glutaraldehyde. Cells whose length is greater than twice the width andwhich possessed one or more pointed extremities under oxygenatedconditions are considered to be irreversibly sickled.

[1149] After various periods of incubation, cells are sedimented at 500g for 5 mm and microvesicles) are isolated from the supernatant solutionby centrifugation at 15,000 g for 15 mm. The microvesicles form a firmbright red pellet sometimes overlain by a pink, flocculent pellet ofghosts (in those cases where lysis was evident) which is removed byaspiration. Quantitation of microvesicles is achieved by resuspension ofthe red pellet in 1 ml of 05% Triton X100 followed by measurement of theoptical density of the clear solution at 550 nm. Optical densitymeasurements at 550 nm give results that are relatively the same asmeasurements of phospholipid and cholesterol content in themicrovesicles. Cell lysis is determined by measurement of the opticaldensity at 550 nm of the clear supernatant solution remaining aftersedimentation of the microvesicles. Larger samples of microvesicles forbiochemical and morphological analysis are prepared from both sickle andnormal cells following incubation of up to 100 ml of cell suspension at37° C. for 24 h in the absence or presence of Ca²⁺. Ghosts are preparedfrom sickle cells after various periods of incubation. The cells arelysed and the ghosts washed in 10 mM Tris HCl buffer, pH 73, containing02 mM EGTA.

[1150] These vesicles are useful as a preventative or therapeuticvaccine as in Examples 15, 16, 18-23, 36.

[1151] Phage Displayed SAgs

[1152] Phages displaying or free tumor homing peptides ligands such asthe tripeptides Arg-Gly-Asp and Asn-Gly-Arg which tripeptides bind tothe integrins avβ3 and avβ5, respectively, that are located on tumormicrovasculature, are conjugated to (1) a SAg peptide, (2) naked DNAencoding a SAg peptide or (3) phage displaying a SAg peptide. Theseconstructs are prepared as in Examples 3 and 5 and are further describedin Jackson R H. et al, In: Protein Engineering: A Practical Approach, A.R. Rees et al (eds), pp. 277-301, Oxford Press, London, 1992. Similarlytumor cells or sickled cells transfected with and expressing SAgs andother molecules given in Tables I and II are also transfected withnucleic acids encoding RGD which facilitates their localization to tumormicrovasculature. These conjugates or transfectants are administeredi.v. and localize to the tripeptides' integrin receptors situated on thetumor microvasculature. Neovascular endothelial cells to which theseconstructs have been targeted are transfected by SAg-encoding DNA sothat they express or secrete SAgs locally. This induces potent local Tcell activation and engender a tumoricidal immune response. Protocolsfor use of such conjugates, i.e., (1) naked SAg DNA conjugated to theintegrin-binding peptides or (2) naked SAg DNA conjugated to phage thatdisplay the integrin-binding peptides, and transfectants in theinduction of anti-tumor immune response are described in Examples 7, 15,16, 18-23, 31

[1153] Nucleic Acid and Nucleoprotein SAg Mimics

[1154] SAgs are often incapable of homing to tumor cells expressing SAgreceptors in vivo because of the existence of naturally occurringSAg-specific antibodies and the affinity of SAgs for class II receptorson a wide variety of cells. To solve this problem, DNA chromatography isused to identify oligonucleotides instead of SAg peptides that bind toSAg receptors which are naturally expressed on tumor cells. The SAgreceptor-specific oligonucleotides are conjugated to a SAg peptide witha functional TCR or NKT cell binding site. Oligonucleotides are alsosubstituted for peptides in the SAg molecule which bind to MHC classreceptors and naturally occurring SAg-specific antibodies. Theseconjugates are used to target SAgs to tumor cells in vivo that eitherendogenously express a SAg receptor or are pre-transfected with nucleicacid encoding a SAg receptor.

[1155] These peptide-oligonucleotide complexes are prepared by chemicalconjugation methods well known in the art. Such receptor specificoligonucleotides may have several fold greater affinity for the SAgreceptor compared to the native SAg. While these peptide-oligonucleotidecomplexes are used predominantly in vivo to target tumor cells bearingSAg receptors, they are also used ex vivo to stimulate T cells to becometumor specific effector cell which are useful for adoptive immunotherapyof cancer (Example 7, 15, 16, 18-23).

[1156] In appropriate recombinant bacteria, nucleic acids encoding theSAg receptor binding site expressed on tumor cells are fused to nucleicacids encoding SAgs. The resultant SAg polypeptide construct consists ofthe amino acid sequence of a SAg and its SAg receptor binding site(which is overexpressed if desired). The SAg with its expressed oroverexpressed SAg binding site is useful in targeting tumor cellsexpressing SAg receptors after administration to a tumor-bearing host.

[1157] In a related construct, the nucleic acid encoding a SAg with anoverexpressed SAg receptor specific binding site is fused to the nucleicacid encoding a native or chimeric SAg with its binding site fornaturally occurring antibodies and its MHC class II binding siteremoved, mutated or replaced by peptides from another SAg against whichthere are no known naturally occurring antibodies. The TCR binding andactivating region of this molecule is conserved. This resulting SAgpolypeptide molecule binds to SAg receptors on tumor cells but alsoretains its capacity to activate the TCR. It is administeredparenterally or orally to a tumor bearing host (orally to a coloncarcinoma patient) and will effectively target tumor cells with SAgreceptors (such as colon carcinoma cells) without being diverted bynaturally occurring antibodies or class II receptor bearing cellspresent in whole blood. As such, this construct is useful in producingan anti-tumor effect when administered to a tumor bearing host as inExample 18-23).

[1158] Using DNA chromatography techniques, nucleic acid specific forSAg receptors on tumor cells are identified. These nucleotides areconjugated to SAg polypeptides which are optionally devoid of class IIbinding sites and naturally occurring antibody binding sites but withconserved TCR binding and activating sites. These constructs are usefulin targeting tumor cells bearing SAg receptors in vivo while retainingSAg amino acid sequences specific for the TCR which are capable ofproducing a tumor specific T cell population effective in adoptiveimmunotherapy of cancer. The selected amino acid sequences are deleted,replaced or added to the SAg molecules using molecular cloning and sitedirected mutagenesis techniques well established in the art.

EXAMPLE 7 General Ex vivo Immunization Methods to Produce Tumor SpecificEffector Cells for Adoptive Immunotherapy of Cancer

[1159] Several days (3 to 60 days) after intratumoral immunization witha nucleic acid construct described herein, tumor draining lymph nodesare removed and placed in tissue culture. These cells are furtherexpanded in vitro with SAg polypeptide for 2-4 days and/or IL-2 in vitrofor a total of 3-15 days. These T cells are then harvested and reinfusedinto the host. T effector cells produced after in vivo immunization withnucleic acid encoding a SAg are expected to display potent anti-tumoractivity.

[1160] Cells transfected ex vivo, are administered to the host whereinthey activate lymphocytes in a number of ways. In one embodiment, theinitial step involves in vivo immunization of hosts using varioustransfectants and constructs as described in Table II. The transfectedcells are introduced into the host tumor, a nearby region,subcutaneously in close proximity to regional lymph nodes, or the lymphnodes draining the tumor. Transfected cells types, constructs and agentsused in this step are given in Table II. Tumor cells are irradiated ortreated with mitomycin C after transfection with nucleic acid encoding aSAg and/or another polypeptide so that polypeptides are expressed andfixed on the cell surface and the tumor cells do not proliferate whenadministered to the host.

[1161] In another embodiment, the initial step involves in vivoimmunization of the tumor bearing host with transfectants, constructsand cells as described in Table III. These agents are administered inclose proximity to the regional lymph nodes with or without a bacterialadjuvant such as bacillus Calmette-Guerin (BCG) or Corynebacteriumparvum. The lymph node cells are harvested 10 days later and tissuecultured for further in vitro immunization/stimulation with SAg or SAgexpressing cells that, optionally, coexpress a tumor associated antigen,costimulatory molecule or antigen presenting molecule.

[1162] Cryopreserved autologous tumor cells for subsequent tumorvaccination and culture are obtained from patients. Fresh resectedtumors are dissociated under sterile conditions into single cellsuspensions by mechanically mincing tumor into 5-mm3 pieces followed byenzymatic digestion. Generally, 1 gm of tumor is digested in a minimumvolume of 40 ml of an enzyme mixture consisting of Hank's balanced saltsolution (HBSS) containing 2.5 units/ml of hyaluronidase type V, 0.5mg/ml of collagenase type IV, and 0.05 mg/ml of deoxyribonuclease type I(all commercially available from Sigma Chemical Co.; St. Louis, Mo.).The digestion is performed at room temperature with constant stirring ina trypsinizing flask for 2 to 6 hours.

[1163] The resulting cell suspension is filtered through a layer of No.100 nylon mesh (Nytek: TETKO, Inc.; Briarcliff Manor, N.Y.) andcryopreserved in 90% human AB serum (GIBCO; Grand Island, N.Y.) plus 10%dimethyl sulfoxide (Sigma) at −178° C. in liquid nitrogen for subsequentimmunization and culture.

[1164] Tumor cells are used in native form, with dinitrophenyl (DNP) orother haptens conjugated to them and then irradiated or treated withcytostatic drugs prior to use. Optionally, the tumor cells aretransfected with nucleic acid encoding a SAg, and/or tumor associatedantigen, and/or antigen presenting molecule, and/or costimulatorymolecule, and/or adhesion molecule, and/or xenogeneic antigen, and/orcarbohydrate modifying enzyme. The nucleic acid is introduced by methodsgiven previously. The cells are then irradiated to a dose of 25 Gy ortreated or with cytostatic drugs, viable cells counted by trypan blueexclusion and the cells resuspended so that a volume of 0.2 to 0.4 mlcontains 1-2×10⁷ with or without ⁷ colony forming units of fresh frozenTICE BCG.

[1165] Patients are vaccinated intradermally (i.d.) at two sitesapproximately 10 cm from superficial inguinal lymph nodes. If necessary,axillary lymph nodes are used. Lymph node regions with previousdissections or clinical evidence of tumor are avoided.

[1166] Accessory cells including DCs, fibroblasts, endothelial cells,monocytes, and macrophages are used after transfection with nucleic acidencoding a tumor associated antigen, and/or SAg, and/or xenogeneicantigen, and/or carbohydrate modifying enzyme. If desired, theseaccessory cells or APCs are transfected with recombinant viral vectorscontaining nucleic acid the encode a SAg, and/or tumor associatedantigen, and/or costimulatory molecule, and/or antigen presentingmolecule, and/or costimulatory molecule, and/or adhesion molecule,and/or xenogeneic antigen. These cells need not be irradiated prior toadministration. These cells are administered using the same cell numbersgiven above with or without BCG.

[1167] Alternatively, patients are vaccinated with various tumorassociated antigens and other agents as described in Table II. Theagents are bound to MHC class I, class II or CD1 receptors or to cellsexpressing these receptors. They are also given alone in doses rangingfrom 0.1 to 10 mg emulsified in various adjuvants well described in theart. A vaccination course includes up to 6 inoculations of the aboveagents at 1-3 week intervals. TABLE III Single Step In Vivo Immunizationof Tumor Bearing Hosts with SAg Nucleic Acids Alone, Combined withNucleic Acid Encoding Other Peptides and SAg Nucleic acids Conjugated toPolypeptides or Liposomes I. Intratumoral injection of nucleic acid 1.Direct injection of SAg nucleic acids into tumor. 2. Direct i.v. orintra-arterial injection of SAg nucleic acids into tumormicrovasculature. a. SAg nucleic acids conjugated to a polypeptideligand specific for a tumor cell, tumor stromal cell, tumormicrovascular or neovascular cell receptors b. Nucleic acid withinliposomes containing a monoclonal antibody. 3. Recombinant virusescontaining nucleic acid. a. Inactivate the virus in the host withgancyclovir II. After in vivo immunization (3-14 days), harvest regionallymph nodes and place in tissue culture. III. Activate and expandlymphocytes. 1. Treat with SAg for 2 days. 2. Treat with IL-2 for 3days. IV. Inject tumor specific effector T cells into host.

[1168] Regional lymph node cells draining tumor sites, lymphoid cellsobtained after the above priming, peripheral blood T cells, and tumorinfiltrating lymphocytes (TILs) are suitable sources of T cells that areactivated to function as effector cells (T cells activated against thecancer cells). T cells are obtained from tumor infiltrating lymphocyteseither before or after tumor vaccine immunization in vivo by the methodsdescribed herein.

[1169] Approximately 10 days after in vivo immunization, an enlargeddraining lymph node is removed and cultured. An immunized lymph nodeused herein is exemplary. A single cell suspension of lymph node cellsis obtained by mechanical dissociation. Briefly, lymph nodes are mincedinto 2 mm³ pieces in cold HBSS with a scalpel. The fragments are thenpressed through a stainless steel mesh with a glass syringe plunger. Theresultant cell suspension is filtered through nylon mesh and washed inHBSS. Cultures are established in 300-ml culture bags (Livecell Flasks;Fenwal, Deerfield, Ill.) with 200 to 250 ml of culture medium (CM: RPMI1640 with 10% human AB serum, 2 mM fresh L-glutamine, 1 mM sodiumpyruvate, 100 mg/ml of streptomycin, and 50 mg/ml of gentamicin all fromGIBCO; Grand Island, N.Y.), containing 1-2×10⁵ lymph node cells/ml and1-4×10⁵ irradiated (60 Gy) tumor cells/ml. Optionally, the lymph nodecells are further separated into populations CD4+CD8+ T cells, NKT cellsand/+T cells. Some SAg complexes are presented bound to MHC class IIreceptors and some such as SAg-LPS complexes or SAg-glycosylceramidecomplexes are presented bound to CD1 receptors either free or on APCcell surfaces.

[1170] After 24 hours, various SAgs or SAg transfected cell types (STCT)given in Table III are added in doses of 10⁵ to 10⁷ cells for 8-72hours. The cells are harvested and used for in vivo administration atthis point. Specific cell populations are selected such as those havinga particular TCR V profile or expressing CD44 using magnetic beads orother separation techniques well known in the art. Optionally, the SAgactivated T cells are expanded. Recombinant IL-2 (Cetus, Emeryville,Calif.: provided by Cancer Treatment Evaluation Program, National CancerInstitute) is added at the initiation of the cultures at a concentrationof 600IU/ml (1 Cetus unit=6 IU of IL-2). Culture bags are incubated at37° C. in humidified 5% CO2. Cell counts from aliquots obtained fromrandom bags are followed to observe lymphoid cell proliferation. Lymphnode cells are harvested when cells reached maximal density, usuallyafter a total of 5-7 days in culture followed by IL-2 at 24 IU/ml for 3days. These intervals are shortened depending on the cell viability,CD44 expression, or V expression or other conditions that adverselyaffect survival, viability, or therapeutic success.

[1171] Antibodies or Fab fragments having specificity for CTLA-4 areadded with or without IL-2 at any point to expand the T cell populationand avert apoptosis. The cells are washed once at the end of STCTincubation and before the addition of IL-2 and/or anti-CTLA-4antibodies. TABLE IV Two Step In Vivo/in Vitro Methods and Agents forProducing Tumor Specific Effector T cells A. In vivo immunization withSAg transfected tumor cells, accessory cells, or virus. 1. Tumor cellstransfected with: a. Nucleic acid encoding a SAg b. Nucleic acidencoding a tumor associated antigen c. Nucleic acid encoding acarbohydrate modifying enzyme 2. Accessory cells transfected with: a.Nucleic acid encoding a SAg b. Nucleic acid encoding a tumor associatedantigen c. Nucleic acid encoding a carbohydrate modifying enzyme d.Nucleic acid encoding an MHC molecule 3. Recombinant viruses containing:a. Nucleic acid encoding a SAg b. Nucleic acid encoding a tumorassociated antigen c. Nucleic acid encoding a carbohydrate modifyingenzyme d. Nucleic acid encoding an MHC molecule B. *In vivo immunizationwith: 1. Irradiated tumor cells. 2. Tumor associated antigens. 3.Irradiated tumor cells conjugated with DNP. 4. Tumor associatedantigen/SAg conjugate or fusion polypeptides. 5. Naked nucleic orplasmid or phage displayed nucleic acid encoding a SAg or attached toliposomes or albumin microspheres. 6. Naked or plasmid or phagedisplayed nucleic acid encoding a SAg/tumor associated antigenpolypeptide conjugate. 7. Tumor cells or accessory cells transfectedwith nucleic acids encoding structures given in Table I Group IA, (pages5 and 6) GM-CSF, IL-2 and other cytokines. (Berns, A J M. et al., HumanGene Therapy 6: 347-368 (1995). 8. Tumor cells transfected with nucleicacids encoding chemokines (T and NKT cell chemoattractants) andgranulocyte chemoattractants (C3a, C5a, MAP). 9. SAg naked DNA fused orin mixture with DNA or structures non-transfected given in Table 1 IA Band C (pages 5 and 6) C. Lymphoid cells from draining lymph nodes areharvested 3-21 days later and placed in tissue culture for furtherstimulation. They are divided into T cell, NKT cell and/T cellpopulations. Alternatively, T cells, NKT cells and/T cells are obtainedfrom the peripheral blood and also placed in tissue culture for furtherstimulation. D. In vitro stimulation of T or NKT cell populations toproduce tumor specific effector cells as described in “C” is carried outwith STCT (“SAg transfected cell types”) or with constructs alone orapplied to appropriate receptors on APCs. MHC class II APCs are used forpresentation of SAg constructs. APCs expressing mannose, or CD1 or CD14receptors are used for presentation of glycosylated SAg, SAg-LPScomplexes, SAg-peptidoglycan complexes or SAg-glycosylceramidecomplexes. Isolated MHC class I, class II, mannose, CD1 or CD14receptors immobilized on solid supports such as polystyrene plates maybe used in place of APCs methods well known in the art. In this formthey bind corresponding ligands in the constructs given above forpresentation to T cells or NKT cells. STCT include tumor cells,accessory cells, antigen presenting cells, prokaryotic cells,autologous, allogeneic or xenogeneic cells lines and viruses. Accessorycells include the following: DCs, monocytes, macrophages, endothelialcells, fibroblasts and NK cells. These cells are transfected withnucleic acids encoding SAgs in combination with the nucleic acids givenbelow. These nucleic acids may include the ISS sequence; SAg genes maybe used with or without the ISS sequence.

[1172] TABLE V Ex vivo Modes of Antigen Presentation to T Cells or NKTCells to Produce Tumor Specific Effector Cells A. Tumor Cells, AccessoryCells, Accessory Cell/Tumor Cell Hybrids, e.g., DC/Tumor Cell)Transfected with:              1. SAg-encoding nucleic acid 2.SAg-encoding nucleic acid and tumor associated antigen nucleic acids (toinclude arrays of tumor associated epitopes) 3. SAg nucleic acid and MHCclass I or II nucleic acids. 4. SAg-encoding nucleic acid andco-stimulatory nucleic acids. 5. SAg-encoding nucleic acid and adhesionmolecule nucleic acids. 6. SAg-encoding nucleic acid andα-galactosyltransferase synthetic nucleic acids or xenogeneic speciesspecific nucleic acids. 7. SAg-encoding nucleic acid and chemoattractantnucleic acids 8. SAg-encoding nucleic acid and glycosylceramidesynthesis nucleic acids 9. SAg nucleic acid and lipopolysaccharidesynthesis nucleic acids 10.  SAg-encoding nucleic acid and microbiallipoprotein or polysaccharide or peptidoglycan membrane or capsularsynthesis nucleic acids 11.  SAg-encoding nucleic acid and SAg receptornucleic acids 12.  SAg-encoding nucleic acid and CD1 receptor synthesisnucleic acids 13.  SAg-encoding nucleic acid and CD14 receptor synthesisnucleic acids 14.  SAg-encoding nucleic acid and SAg promoter and/orglobal regulator nucleic acids 15.  SAg-encoding nucleic acid andoncogene and/or transcription factor nucleic acids 16.  SAg-encodingnucleic acid and angiogenesis factor or receptor nucleic acids 17. SAg-encoding nucleic acid and growth factor receptor nucleic acids 18. SAg-encoding nucleic acid and cell cycle protein nucleic acids 19. SAg-encoding nucleic acid and heat shock protein nucleic acids 20. SAg-encoding nucleic acid and chemokine nucleic acids 21.  SAg-encodingnucleic acid and cytokine nucleic acids 22.  SAg-encoding nucleic acidand tumor suppressor nucleic acids 23.  SAg-encoding nucleic acid andantigen processing and trafficking nucleic acids B. Additional in vitroStimulatory Agents (preferred receptor) 1. Tumor peptides (Class I orClass II) 2. Tumor peptide-SAg conjugates or fusion proteins (Class I orClass II). 3. Lipopolysaccharide-SAg conjugate (Class II or CD14) a.arabinose b. mycolic acid c. teichoic acid d. muramic acid(Staphylococcal cell wall glycoprotein) e. mannan proteoglycans f.chondroitin-sulfate 4. Glycosylated SAgs. (Class II or mannose) 5.SAg-glycosylceramide conjugates (class II or CD1) a. GalCer conjugate b.Gal conjugate 6. SAg-proteosome conjugates 7. SAg or glycosylated SAg orSAg-glycosylceramide conjugates or SAg-lipopolysaccharide or SAg-peptidoglycan conjugates coupled to proteosomes 8. SAg or glycosylatedSAg or SAg-glycosylceramide conjugates or SAg-lipopolysaccharideconjugates or SAg-peptidoglycan conjugates expressed on or coupled toliposomes 9. Conjugates having a Superantigen component (polypeptide ornucleic acid) and a partner that is either a single component or aconjugate of 2 or more components (protein, carbohydrate, lipid DNA) asindicated below. Superantigen (Protein or DNA) Partner (Single Componentor Conjugate) 1. DNA coding sequence 2. Polypeptide 3. Nucleic acid 4.Tumor associated Peptide 5. Tumor Antigen-MHC protein 6. LPS 7.Lipoarabinomannan 8. Ganglioside 9. Glycosphingolipid 10.Ganglioside-CD1 receptor 11. Glycosphingolipid-CD1 receptor 12.Glycosylceramide (e.g., Gal-Cer) 13. GalCer-CD1 receptor 14. Gal 15.Arg-Gly-Asp or Asn-Gly-Arg 16. iNOS 17. Gb2 or Gb3 or Gb4 18. (Gb2 orGb3 or Gb4)-CD1 receptor 19. -GPI-(Gb2 or Gb3 or Gb4) 20. -GPI-(Gb2 orGb3 or Gb4)-CD1 receptor 21. Vero toxin 22. Vero toxin A or B Subunit23. IFNα receptor peptide homologous to VT 24. CD 19 peptide homologousto VT 25. LDL, VLDL, HDL, IDL 26. Apolipoproteins (e.g., Lp(a),apoB-100, apoB-48, apoE) 27. OxyLDL, oxyLDL mimics, (e.g.,7β-hydroperoxycholesterol, 7β-hydroxycholesterol, 7- ketocholesterol,5α-6α-epoxycholesterol, 7β-hydroperoxy-choles-5-en-3 β-ol,4-hydroxynonenal (4-HNE), 9-HODE, 13-HODE and cholesterol-9-HODE) 28.OxyLDL by products (e.g. lysolecithin, lysophosphatidylcholine,malondialdehyde, 4- hydroxynonenal) 29. LDL & oxyLDL receptors (e.g.,LDL oxyLDL, acetyl-LDL, VLDL, LRP, CD36, SREC, LOX-1, macrophagescavenger receptors) 30. phytosphingosine, -GPI-phytosphinosine 31.tumor associated lipid antigens 32. glycolipid, proteolipid,glycosphingolipid, sphingolipid with inositolphosphate-containing headgroups, phytoglycolipids, mycoglycolipids, -GPI-sphingosines orGPI-lipids 33. sphingolipids with inositolphosphate-containing headgroups having the general structure: ceramide-P-myoinositol-X with Xreferring to polar substituents comprising ceramide-p-inositol- mannose,inositol-1-P-(6)mannose(αl,2inositol-1P-(1)ceramide,(inositol-P)2-ceramide, inositol-P- inositol-P-ceramide,inositol-P-inositol-P-ceramide. 34. tumor associated glycan antigensconsisting of peptidoglycans or glycan phosphotidyinositol (GPI)structures C. STCT or SAg-tumor peptide conjugates are incubated with invivo immunized T cells or NKT cells for 2-4 days and then with IL-2 for2-5 days. D. The tumor specific effector cells are then harvested andinjected in doses of 10¹⁰-10¹² every 3-7 days for 1-6 treatments. E.Viruses are transfected into tumor cells, accessory cells, antigenpresenting cells, allogeneic or xenogeneic cells. They arepre-programmed with DNA for SAgs alone or in combination with genesgiven in D. They may also utilize the host genome to produce a new geneproduct as for example the host -galactosyltransferase. Viruses mayinclude the following: 1. Adenoviruses. 2. Vaccinia virus. 3. Equineencephalitis virus. 4. Influenza virus. F. In an additional method,tumor associated antigens are bound to MHC class I positive cells andused to activate T cells. SAg-lipopolysaccharide complexes andSAg-glycosylceramide complexes are bound to CD1 or class II receptors onAPCs. In addition, SAg-lipopolysaccharide complexes orSAg-glycosylceramide complexes are presented bound to class II positiveAPCs. Alternatively, unbound tumor associated antigen/SAg. conjugates orfusion products are added at a 0.1 to 200 mg/ml dose for 2 days. This isfollowed by STCT incubation or by native or mutant SAg treatment for 2days.

[1173] For comparative analysis, peripheral blood lymphocytes (PBL) areobtained from patients the same day as the lymph node harvest. PBL areisolated by Ficoll-Hypaque gradients from 60-ml of heparinized bloodsamples. The PBL are placed in culture utilizing 24-well tissue cultureplates at the same cell density as lymph node cells. PBL are harvestedat maximal cell density and characterized by phenotype analysis andcytotoxicity. T cells, NKT cells, and NK cells are isolated by wellknown methods described in the art (Colligan, J E et al., eds, CurrentProtocols in Immunology, John Wiley, New York, 1996).

[1174] PBL are separated by Ficoll/hypaque sedimentation. Cells arerecovered from the interface, washed in PBS, and pelleted. Peripheralblood mononuclear cells enriched for MHC class I molecules or MHC classII molecules are used to bind tumor associated antigens or tumorassociated antigen/SAg conjugates for in vitro or in vivo immunization.

[1175] Cryopreserved groups of autologous PBMCs are thawed, washed twicein PBS, resuspended at 5 to 8×10⁶ cells/ml in CM and pulsed with 1 mg/mlpeptide in 15 ml conical tubes (5 ml/tube) for 3 hours at 37° C. ThesePBMC stimulators are then irradiated at 3000 rads, washed once in PBS,and added to the responder cells at responder stimulator ratios rangingbetween 1:3 and 1:19.

[1176] Tumor infiltrating lymphocytes are isolated from fresh surgicalbiopsies. Briefly, tumor tissues are minced into 1-mm3 pieces that arethen dissociated into single cell suspensions in Dulbecco's modifiedminimum essential medium (Gibco; Grand Island, N.Y.) supplemented with10% heat-inactivated human AB serum (NABI, Miami, Fla.), 0.05%collagenase (type 4; Sigma Chemical Co., St. Louis, Mo.), and 0.002%DNase (type 1; Sigma) on a magnetic stirrer for 1 hour. Subsequently,the tissue digests are washed and passed through a nylon mesh and tumorinfiltrating lymphocytes and tumor cells are separated on discontinuous(75%/100%) Ficoll/Hypaque gradients.

[1177] Lymph node lymphocytes are obtained by mechanical dissociation oftissues, followed by washing in medium and centrifugation onFicoll/Hypaque gradient [Newell K A, et al., Proc. Natl. Acad. Sci. USA,88:1074 (1991)]. Cryopreserved suspensions of tumor cells/tumorinfiltrating lymphocytes are defrosted, washed, and separated byallowing tumor cells to adhere to the surface of plastic wells. Therecovered non-adherent tumor infiltrating lymphocytes are transferred to6-well plates and cultured in serum-free AJM-V medium (Gibco)supplemented with 6,000 U/ml of IL-2 (Cetus-Chiron, Emeryville, Calif.)for 8 days. Tumor cells are cultured as adherent monolayers inDulbecco's modified Eagle's medium (DMEM, Gibco) supplemented with 10%(v/v) of fetal calf serum. Any activated lymphocytes can be used in themethod given above. In a preferred embodiment, lymphocytes expressing apredominant TCR V phenotype in tumor tissue or peripheral blood beforeor after treatment are isolated and expanded by standard procedures.

[1178] Antibodies to various TCR Vβ subsets are immobilized on inertsolid supports and incubated with blood cells and/or tissue cells toinclude bone marrow and peripheral blood or lymphoid tissue cells andtumor infiltrating lymphocytes.

[1179] The bound T cells are eluted with various buffers. Suitablebiocompatible inert supports include polystyrene, polyacrylamide, nylon,silica, and charcoal as well as others known in the art. The supportsare derivatized for covalent binding of antibodies with agents wellknown in the art including heterobifunctional compounds, carbodiimide,and glutaraldehyde. The enriched population of V-bearing T cells is thenused for in vitro immunization with a SAg in native or mutant formcapable of activating the dominant TCR Vβ bearing lymphocytes. IL-2 isused to further expand the cell population as described above.

[1180] Effector lymphocytes obtained after in vivo sensitization arestimulated in vitro with tumor associated antigens bound to irradiatedPBMC (which act as stimulator cells) for 8-72 hours. DCs, macrophages,or other class I-bearing cells are used to present the tumor associatedantigens. The T cells are then analyzed for TCR Vβ and/or CD44expression. An STCT expressing a SAg is then added to the culture (1picogram to 10 microgram). If a given V predominance is noted afterantigen stimulation, then an STCT or SAg known for its ability tospecifically stimulate that Vβ subset is selected for use in activation.Culture proceeds for 18-72 hours. The TCR Vβ and CD44 profile ofstimulated T cells are then rechecked. IL-2 (12-25 IU) and/oranti-CTLA-4 antibodies are added for an additional 8-72 hours afterwhich the cells are harvested for use. The optimal timing of STCTintroduction after tumor antigen stimulation is between 3 and 14 days.

[1181] Antigen-presenting cells (APCs) of all kinds such as DCs, B cellsor macrophages with appropriate MHC class II molecule binding sites forsoluble SAgs are used or the SAgs are presented alone or in immobilizedform without APCs. Optionally, STCTs are used without APCs. Before IL-2administration, effector cells are re-stimulated weekly by washing andreplating in 24 well plates at a concentration of 2.5×10⁵ cells/ml inCM. This is continued for 3-10 cycles until enough cells are availablefor IL-2 expansion. T cells are cloned 7 days after the several cyclesof stimulation in 96-well round bottom plates at 0.3 cells/well with5×10⁴ stimulator tumor antigen-PBMC, SAg, or STCT and 25-50U recombinantIL-2 in a volume of 200 ml.

[1182] For long term growth, clones are transferred to 24 well platesand 10⁶ cells/well and stimulated weekly with SAg or STCT plus optimally5×10⁵ tumor associated antigen-PBMC and 25-50U/ml of IL-2. After clonesgrow to greater than 2×10⁶ cells, the clones are maintained by culturingwith STCT only for 48 hours, washing to remove STCT, and replating infresh media for 5-7 days with 25-50 U/ml IL-2.

[1183] The initial incubation is with the selected tumor associatedantigen such as MART-1 for 1-3 days with the latter reagents followed byV profiling and re-stimulation with SAg by methods given above. TheMART-1 is presented attached to HLA-A1⁺ cells of PBMC. Cytotoxicactivity is tested after the first and/or second rounds of sequentialstimulation with tumor associated antigen and SAg given below.

[1184] The tumor-specific effector T cell population is immortalized astumor specific T cell hybridomas. These hybridomas are generated byimmunization in vitro of human T cells as described herein. The expandedT cells are then fused to a thymoma and cloned by limiting dilution orother methods well known in the art. Cells are cultured in completetumor medium composed of Eagle's minimal essential medium supplementedwith 10 mM 2-mercaptoethanol, 10% fetal calf serum, 10% Mishell-DuttonNutrient cocktail, 100 U/ml penicillin G, and 200 mg/ml streptomycinsulfate. Other well known culture media can also be used.

[1185] For SAg immunization in vitro, various antigen presenting cellsare used including MHC class II-positive T cells as well as thoseexpressing CD1. Purified MHC class II or CD1 molecules alone orimmobilized are substituted for APCs in some cases. Moreover, T cellsare activated by some SAgs without APCs when presented to T cells inimmobilized form or in the presence of various cytokines such as IL-1,IL-2, IL-4, or IL-6 or xenogeneic antigens. Various costimulants such asB7-1 and B7-2, adhesion molecules such as ICAM-1 and VCAM-1, or GalCerare used together with SAgs and MHC class II positive APCs orimmobilized MHC class II peptides to augment the T cell or NKT cellresponse.

[1186] Tumor associated antigen immunization is also involved in thebinding of peptides to MHC class I bearing APCs of multiple origins.Various cytokines including, but not limited to, IL-1, IL-2, IL-4,IL-12, or LPS are used in vitro or in vivo to expand the antigenspecific clone of T cells and avert the development of T cell anergy.

[1187] Specialized Forms of Tumor Specific Effector Cells and Hybridomas

[1188] Tumor specific T or NKT cells with TCR V and/or CD44 selectivityare produced by transfecting uncommitted stem cells with nucleic acidsencoding particular TCR V chains. Likewise, a T cell cloneoverexpressing CD44 is produced by transfecting T cells with nucleicacids encoding CD44. A hybridoma expressing a tumor associated antigenwith a dominant TCR V phenotype or CD44 expression is produced in thisway. Such a T cell hybridoma or cell line is stimulated exogenously by aSAg or a SAg mutant with a TCR V or CD44 selectivity corresponding tothat expressed predominantly by the T cell hybridoma. The result is aclone of tumor specific T cells capable of being expanded by exposure toSAg in vitro or in vivo.

[1189] CD44 expression is induced in a T cell, NKT cell or TCR/T cellpopulation after activation in vitro or in vivo with SAgs alone ortogether with any of the T or NKT cell stimulating constructs andmethods described herein. The in vivo and in vitro activation steps andimmunization protocols are given in Examples 7, 15, 16. 18-23. The CD44positive T cell population exhibits upregulated primary adhesionproperties and is capable of effectively trafficking and homing to tumorcells in vivo and particularly to sites of SAg (in native or nucleicacid form) injection i.e. tumor Nucleic acids encoding CD44 or acarbohydrate modifying agent will induce CD44 expression on the T cellsurface. A preferred in vivo method of use involves intratumoralinjection of SAg DNA into tumor sites which induces expression of CD44on T cells resulting in enhanced T cell trafficking to the site of SAgadministration.

[1190] T cells are genetically engineered to overexpress CD44 after SAgstimulation. This is accomplished by transfection of T cells or NKTcells with nucleic acid encoding CD44 as well as nucleic acids encodingglycosyltransferases. This results in the overexpression of CD44upregulation of the adhesive properties of CD44. Such CD44 enrichedclones are harvested after SAg stimulation, enriched, and administeredfor adoptive immunotherapy of cancer (Examples 6, 7, 15, 16, 18-23).

[1191] Additionally, T or NKT cell clones or hybridomas are producedwhich express a chimeric TCR consisting of an invariant chain withspecificity for GalCer or and a chain that binds a SAg. The V regionwhich is specific for the SAg is overexpressed on the TCR permittinggreater responsiveness to exogenous SAg. This chimeric TCR recognizesand is stimulated by an exogenous SAg with a TCR V selectivitycorresponding to the predominant TCR V phenotype of the T or NKT cell.Such T or NKT cell lines are cloned and hybridomas produced by methodswell known in the art.(Current Protocols in Immunology, pp. 7.21.-7.21.9John Wiley, New York, 1991) The expanded clone of tumor specific T cellsproduced in this way is useful for adoptive immunotherapy of cancer bymethods given in Examples 7. 15, 16, 18-23.

[1192] T cell clones are produced due to asynchronous TCR V locusrearrangements at low but significant frequency in which both TCR Vsegments are part of two functional TCRs. Such clones are produced fromuncommitted stem cells in which nucleic acid encoding two chains aretransfected, one having specificity for a tumor associated antigen andanother having SAg specificity. Hence, a clone of T cells with dual VTCR expression is produced which is capable of reacting with a tumorspecific and a SAg. This clone is expanded by binding either or bothligands. These expanded clones consisting of tumor specific effector Tcells are used for adoptive immunotherapy of cancer by protocols givenin Examples 7, 15, 16, 18-23).

[1193] T cells or NKT cells clones or hybridomas expressing TCR Vα andVβ chains with specificity for GalCer and SAg, respectively, areproduced by fusion of NKT cell DNA encoding the GalCer and SAg receptorswith DNA from an appropriate thymoma. This GalCer receptor and SAgreceptors are expressed on the α and β chains of the TCR, respectively.Upon exposure to GalCer or SAg, these cells are further activated toexpress CD44 which enhances their homing and adhesive properties. NKT orT cells expressing high levels of IFN, GM-CSF, and IL-10 are selectedand cloned. The clone of T cells producing IFN and expressing GalCer,SAg and CD44 is then expanded and immortalized. With its properties oftumor recognition, SAg and glycosylceramide activation, IFN productionand effective in vivo trafficking, this T or NKT effector cellpopulation is preferred for adoptive immunotherapy of cancer by methodsgiven in Examples 7, 15, 16, 18-23).

[1194] Additional measures to avert apoptosis and augment proliferationcapacity in SAg activated T cells include the use of anti-CD28antibodies and inhibition of CTLA-4 on T cells. CTLA-4 on T cells isblocked by specific antibodies or fragments.

[1195] Alternatively, a T cell clone is used in which CTLA-4 isgenetically deleted. When stimulated by

[1196] SAg, these cells proliferate to a greater extent compared to SAgalone. Cell populations in which CTLA-4 is deleted or blocked areselected to have a predominant V bearing lymphocyte population that isactivated after in vivo or ex vivo tumor associated antigen stimulation.After CTLA-4 deletion or blockade, the appropriate SAg with Vβselectivity is chosen to expand this population. To avert uncontrolledproliferation in vivo, the thymidine kinase gene of the HSV isco-transfected to enable elimination of these cells in vivo if desired.

[1197] Measures to produce an effector T cell population with anoverexpressed TCR V and/or V chains specific for a given SAg involve thetransfection of nucleic acids encoding the desired V or V regions into Tcells as in Example 1. To lower the activation threshold of the T cellor NKT cells to SAg or SAg-tumor peptide-MHC or CD1, the T cell or NKTcells are transfected with nucleic acid encoding a tyrosine kinase orother signal transduction initiating molecules which can dimerize in themembrane with the TCR tyrosine kinases thereby lowering the thresholdfor activating the signal transduction pathway. The deletion of thesignal transduction inhibitory region of the TCR to produce sustainedsignal transduction is done by site directed mutagenesis as in Example24.

[1198] The T cell population used for adoptive immunotherapy afterstimulation with SAg are enriched for interferon production using anIFN-gamma capture assay as described by Becker et. al. Nature Med. 7:1159-1162 (2001). Such enriched IFN gamma secreting T cells have beenshown to be cytolytic and optimal for adoptive transfer. (See Example 66for methods of production and use of the IFN gamma secreting T cell inadoptive immunotherapy of cancer).

[1199] In an additional embodiment, to avert apoptosis of T cells afterex vivo superantigen induced stimulation, they are coincubated withcytokines IL-2, IL-4, IL-7 and IL-15 on in vitro. IL-4 and IL-15 appearsto exceed IL-2 and IL-7 in their T cell sparing effects. The Tcell-cytokine cultlure conditions are given in Vella A T et al., ProcNatl Acad Sci 95:3810-5 (1998).

[1200] In an additional embodiment. CD25 positive T cells are alsoactivated by SEs in vitro and in vivo (Rosendahl A et al., Infect Immun5:5118-24 (1997) and inhibit the activity of the tumor cytotoxic Tcells. Depletion of CD25+ T cells with a single injection of 0.4 mg ofanti-CD25-depleting antibody resulted in an anti-tumor effect (SutmullerR P M et al., J. Exp. Med. 194: 823-832 (2001). Accordingly, T cells foruse in adoptive therapy are depleted of CD25+ T cells in vitro usinganti-CD25 antibodies in quantity sufficient to reduce the CD25population to undetectable levels. These antibodies are introduced 5 to72 hours after stimulation to the T cells with superantigen. The CD25depeted T cell population is then expanded in IL-2 (and/or othercytokines shown to be optimal for sparing T cells from apoptosis asgiven in Vella A T et al., supra and previous patents and patentapplication in this line), collected and administered to the patient.

EXAMPLE 8 Prevention of Anergy in T or NKT Tumor Specific Effector Cells

[1201] The SAg stimulated tumor specific effector T cells used foradoptive immunotherapy of cancer may not function when infused unlessmeasures are taken to prevent T cell anergy or activation-induced celldeath (AICD) by interdicting the Fas mediated pathway. The Fas ligand(FasL) has been identified as a type II transmembrane polypeptide of theTNF family. These two related receptor-ligand systems signal apoptosisthrough closely related but distinct pathways. T cell phenotypes thathave diminished expression of Fas or FasL show delayed anergy inductionand shortened periods of non-reactivity compared to Fas-expressingcells. Activation-induced cell death (AICD) induced by SAgs in vitro orin vivo is averted using Fas-deficient T cells, including those withdown-regulated Fas or FasL receptors as well as those with masked orblocked Fas receptors. A Fas-IgG fusion protein is added during the SAgactivation phase to prevent AICD or anergy induction. Measures such asthose above (or by treating with anti-CTLA-4 antibodies or activation ofCD28 before, during, or after STCT stimulation) protect T cells fromanergy or AICD. In this way, these manipulations prolong T cell survivalin vitro and enhance tumoricidal activity in vivo after the T cells areactivated by tumor associated antigen plus SAg or tumor associatedantigen-SAg conjugates in vitro.

[1202] SAg nucleotide alone or fused to tumor peptide nucleotide may befurther fused with an antisense nucleotide capable of inhibiting theapoptosis pathway.

[1203] When expressed in T cells, this combination of genes wouldpromote the generation of tumor specific effector T cells which would beresistant to AICD. Oligonucleotide antisense molecules that inhibit keysteps leading to apoptosis may be fused to SAg DNA in order to preventthe T cells from undergoing AICD. SAg DNA may also be fused with themulti-drug resistance (MDR) gene to make the T cells refractory tochemotherapeutic agents and sensitive to anti-apoptosis drugs. Certaindrugs or radiation may be used together with SAg DNA for additive orsynergistic inhibition of the apoptosis pathway in the doubly ormultiply transfected T cells.

[1204] SAg DNA may also be linked operatively to promoter genes such asthose inducible by corticosteroids or heavy metals (e.g., themetallothionein promoter) and regulatory DNA sequences that act as Tcell on/off sensors responsive to exogenous cytokines, inflammatorystimuli and changing external conditions such as oxygen tension and pH.A particular advantage of SAg DNA is that its expression will promote Vreceptor downregulation and internalization so that these receptors areunavailable to exogenous SAg. SAg DNA is modified in several ways tointroduce protein binding sites for key transcriptional elements whichmay inhibit apoptosis. Insertion of such sites at the bending domains ofSAg oligonucleotides renders them capable of inducing key TH-1 cytokinesand cell proliferation while averting AICD.

[1205] SAg DNA is also capable of reversing the T cell anergy andsignaling defect which may be localized to the chain in cancer patients.This is accomplished by providing transcriptional binding sites on theSAg DNA which bypass the conventional chain activating signals and thepathway to IFN and IL-2 production. In the same way, SAg DNA alsobypasses the defective signal by activating a complex that containsSTAT-1 which binds a GAS-like palindromic sequence located in the IFNresponse region of the FCRI gene. Such anergy in T cells may also bereversed by alternate cytoplasmic tails that are activated by SAgbinding to the TCR Vα and Vβ chains. Moreover, nucleic acid encodingProtein A and especially domain D (that binds to the Ig VH3 region) maybe fused to SAg DNA in order to bring about activation of the IL-2 andIFN genes that resulting in T cell proliferation and IFN productioncoupled with up-regulated surface receptors for the Ig VH3 domain.

[1206] Anergy in SAg-activated tumor-specific T or NKT effector cells(or hybridomas) is known to be averted by in vitro or in vivoco-administration of IL-2, IL-1, LPS and tumor specific peptidesspecifically interfere with SAg driven anergy.

[1207] Methods and doses for use of these agents with SAg activated T orNKT cells are given in Examples 7, 15, 16, 18-23.

[1208] Tumor specific T or NKT effector cells or hybridomas prepared byvarious methods described above are administered according to theadoptive therapy protocol of Examples 7, 15, 16, 18-23 (the preferredmethod). The experimental tumor models and human cancers for which theanti-cancer efficacy of these cells can be demonstrated are provided inExample 16.

EXAMPLE 9 Reactivation of Anergized Tumor-Specific T or NKT Cells by SAgand SAg Receptors

[1209] Preferred tumor-specific effector cells for adoptiveimmunotherapy of cancer are autologous T cells. However, in the courseof tumor growth, T cells become anergized to the host's own tumor andare incapable of an adequate immune response to the tumor. DampenedTCR-triggered responses are caused by suppression of effector moleculesthat couple cell surface receptors to early and late intracellularsignaling events. For example, basal and induced tyrosinephosphorylation of many signaling proteins is reduced due to deficits atmultiple points, including the inositol phosphatase pathway. Thisdown-regulates cytokine production and decreases nuclear transcriptionfactors of TH1 helper cells.

[1210] Two functionally distinct signal transduction pathways arecoupled to the TCR. Native or mutant SAgs activate anergic T cells viaan alternate pathway without the conventional increases in Ca++mobilization or detectable phosphatidylinositol hydrolysis that followligation of the TCR by peptide/MHC complexes. Native, mutant orderivatized SAgs are administered to stimulate anergized T and/or NKTcells to become tumor-specific effector cells now fully reactive againsttumors. Such cells are-used also in adoptive immunotherapy of cancer asdescribed in Examples 7, 15, 16, 18-23. Nucleic acid constructscomprising DNA encoding SAg and SAg receptor are provided to reverse Tcell anergy in cancer patients.

[1211] Anergic T (or NKT) cells transfected with DNA encoding a SAgreceptor express the receptor on the cell surface. Binding of exogenousSAg to this receptor generates T cell activating signals, so that theactivated T cells can be used for adoptive immunotherapy.

[1212] DNA encoding a SAg peptide is transfected into cancer patients'anergized T and/or NKT cells. These DNA constructs also contain the ISS(described above). The T and/or NKT cell transfectants have revitalizedproliferative activity when stimulated by tumor-specific antigen andexogenous SAg. The cells are used for adoptive immunotherapy of cancer(Examples 7, 15,16, 18-23).

[1213] Anergic T cells from cancer patients are transfected in vivo orin vitro, resulting in a population of tumor reactive effector T cellsin vivo or ex vivo. The ex vivo transfected T cells are used foradoptive immune therapy as described in Example 15, 16, 18-23. In thecase where SAg receptor is expressed by transfected T cells, these cellsare activated by locally or systemically by SAgs to result intumor-specific effector cells.

[1214] Additional manipulations that assist in restoring responsivenessto anergic T cells include removal of the (T or NKT) cells from theimmunosuppressive microenvironment and transfer into tissue culture fora short period before stimulation with SAg. Furthermore, defectivesignaling in patient T or NKT cells may be reconstituted by transfectionwith DNA encoding CD3-2 or fyn that is either in a single constructwith, or cotransfected with, DNA encoding SAg and/or SAg receptor. Now,surface activation of the SAg receptor triggers CD3− signaling and Tcell proliferation.

[1215] SAg activation of a T cell surface ganglioside (such as GD3) isalso used to reverse T cell anergy in cancer patients. SAg coupled toGalCer, lipopolysaccharides or proteosomes are even more effective inactivating such anergized T cells. Coordinate activation of CD69 withphorbol esters in combination with SAg stimulation also reverses T cellanergy.

EXAMPLE 10 Tumor Specific Effector T or NKT Cells as Lymphoid Organoids

[1216] Tumor specific T and/or NKT effector cells (or hybridomas withsuch cells) are prepared ex vivo in the form of a lymphoid organoid andimplanted into tumor-bearing hosts. The organoid consists of the tumorspecific lymphocytes either activated by SAgs, transfected to expressSAg alone or in combination with the other proteins or anti-tumormoieties described herein. The cells are encased in semi-permeablemembranes that allow for their progressive entry into the blood andlymphatics after implantation into the host. Such organoids areimplanted preferentially at sites adjacent to lymphatics or bloodvessels that drain organs or regions of known tumor involvement.However, they may also be implanted subcutaneously, intraperitoneally inaddition to intra-tumorally or adjacent to a tumor site. The advantageof the organoid is that it continuously provides proliferatingtumor-specific effector cells that recognize traffic to tumor sites in aphysiological manner. This approach avoids negative selection,functional deficiencies and storage problems associated with long termcultured cells.

[1217] Organoids are encased in macrocapsules, sheaths, rods, discs, orspherical dispersions or microcapsules. Microcapsules are made ofhydrogels such as polysaccharide alginate that are optionally coatedwith polyanions and again with alginate. Macrocapsule and vasculardevices consists of acrylonitrile-vinyl chloride copolymers or cellulosenitrate membranes. In one approach, scaffolds composed of syntheticpolymers serve as cell transplant devices. The polymers are degradableor non-degradable materials that disappear from the body after theyperform their function to obviate concerns about long-termbiocompatibility.

[1218] These devices serve as structural and functional tissue units bythe transplanted cells. The open system implants are designed so thatthe polymer scaffold guides cell organization and growth and allowsdiffusion of nutrients and cells. The cell polymer matrix ispre-vascularized or becomes vascularized as the cell mass expands afterimplantation. Vascularization is induced naturally by the host orartificially by secretion of angiogenic factors from host cells.Optionally, the angiogenic proteins are genetically engineered into thehost T cells in vitro before implantation or in vivo before or afterimplantation.

[1219] To maintain or facilitate targeting of the cells to tumors orinvolved organs, the lymphocytes are transfected with DNA encodingpolypeptides that enhance homing and trafficking ability to the sites oftumor burden (e.g., brain, liver, lung). The organoid lymphocytesexpress no CTLA so that they may proliferate (in vitro and in vivo)without the need for exogenous IL2. Alternatively, cells are transformedto express herpes simplex virus thymidine kinase, making themsusceptible to killing by gancyclovir. This curtails uncontrolledproliferation caused by the CTLA-4 deletion (or inhibition). Exogenouscontrol of antitumor activity is achieved through the use of induciblepromoters, such as those responsive corticosteroids or metals.

EXAMPLE 11 Tumor Specific Effector Cells or Tumor Cells ExpressingProtein A, Protein A Domains and/or Angiostatin

[1220] It is desirable to express Fc receptors (FcR) or Ig VH₃ domainson tumor cells to promote binding by immunoglobulins and enhance damageby antibody dependent cellular cytotoxicity. By introducingStaphylococcal Protein A, or its domains A-D into tumor cells whichoverexpress FcR and VH3 the tumor cells bind immunoglobulins (includingthose with α-gal specificity). Signaling of T cells occurs via highaffinity binding to FcR (FcRI) of protein A-IgG complexes; such bindingbypasses the CD3− blockade in tumor bearing patients. These transfectedtumor cells are useful as a vaccine. Likewise, nucleic acids encodingprotein A and its domains A-D are transfected into partially or fullyanergized T or NKT cells of cancer patients. Exogenous immunoglobulinsstimulate the generation of tumor-specific effector T or NKT cells whichare used in adoptive immunotherapy (Examples 7, 15, 16, 18-23).

[1221] DNA encoding Staphylococcal protein A and its domain D areco-transfected into these tumor cells resulting in the joint surfaceexpression of: (1) protein A and FcR to which it binds and/or (2) domainD and Ig VH3 to which it binds.

[1222] When DNA encoding protein A or domain D, fused to a signalsequences that route and anchor the protein A peptide to the tumor cellsurface, is introduced into tumor cells, such tumor cells are excellenttargets for parenterally administered SAg polypeptides (particularlythose for which no natural antibodies exist). Tumor cells expressingprotein A and domain D and also expressing FcRs on the cell surface,have heightened sensitivity to complement mediated lysis.

[1223] Tumor cells cotransfected to express protein A and Gal (byintroduction of the appropriate glycosyltransferase) are capable ofreacting with natural anti-Gal antibodies, Ig Fc fragments and Ig VH3domains, which stimulate an enhanced tumoricidal response.

[1224] Angiostatin is a circulating angiogenesis inhibitor which is38-kDa internal fragment of (mouse) plasminogen that contains the firstfour disulfide-linked kringle domains. In vivo, angiostatin suppressesneovascularization in several traditional assays (chick chorioallantoicmembrane assay and mouse corneal assay). Proteases released by tumorcells cleave circulating plasminogen to generate angiostatin.Metalloelastase produced by tumor infiltrating macrophages generatedangiostatin production by murine Lewis lung carcinoma. In the presentinvention, nucleic acid encoding angiostatin (Cao Y et al., J. Clin.Invest. 101: 1055-1063, (1998)) are cotransfected into tumor cells withnucleic acid encoding SAg (as in Example 1). The tumor cellcotransfectants express and secrete SAg and angiostatin. Such cells areused directly as a preventative vaccine (Example 8) or as a therapeuticvaccine to treat established tumor including micrometastases. Methodsfor using these cells in vivo are in Examples 7, 12, 16, 18-23.

[1225] In addition, tumor cells are cotransfected to express angiostatinand protein A (and/or its domains). Any nucleic acid construct shown inTable I may also be used in combination to transfect tumor cellstogether with protein A, its domains and angiostatin.

EXAMPLE 12 SAg Receptor

[1226] Colon carcinoma is used as the tissue source for the SEBreceptor. Mixtures of different detergents at low concentrations areused. The protocol for screening detergents for solubilization of MAChRsis readily adaptable to other receptor types. The membranes aresuspended for 5-10 min at pH 7.5, 20 mM Tris-HCl, pH 7.5, or 20 mMsodium phosphate, pH 7.0-7.5. For screening purposes it is unnecessaryto add complex proteolysis inhibitor cocktails. The presence of EDTA (1mM) to inhibit calcium-activated proteases and of PMSF or benzamidine(0.1 mM) to inhibit serine proteases is sufficient. Mg2++ (2 mM) isadded. The membranes are prelabelled with a radioligand in the presenceand absence of a suitable unlabelled ligand to determine the total andnon-specific binding. Non-specific binding is subtracted from totalbinding to obtain the specific binding. A high enough concentration oflabeled ligand to saturate the binding site (10×Kd) is used, so that thebinding capacity is measured. The unlabelled ligand is used at aconcentration of 1000×Kd. The normal criteria for specific binding mustbe fulfilled. The incubation is sufficient to reach equilibrium.Prelabelled membrane suspension (0.5 ml) is added to a series ofcentrifuge tubes a 4° C. An equal volume of detergent solution in thesame buffer is added to obtain a series of different final detergentconcentrations, e.g., 0, 0.1, 0.2, 0.5, 1.0, 2.0% w/v.

[1227] The tubes are mixed and incubated for 60 min. at 4° C.Solubilization is assisted by stirring or mixing, e.g., with arotating-wheel end-over-end mixer. The tubes are centrifuged for 30-60min at 100,000×g for 60 min. For screening, a lower speed spin, e.g.,10,000×g for 5 min (such as in a microfuge) is acceptable. Supernatant,0.2 ml, is applied to a 2 ml column of Sephadex G50 equilibrated withthe selected detergent at 0.1%. When the sample has run in, 2 x 0.2 mlof detergent-buffer is applied and then the void volume fraction iseluted with 0.5 ml of detergent buffer. This procedure is carried out,the remaining material is removed, and 10 ml of aqueous scintillationcocktail is added and the radioactivity counted. Sephadex G50 issubstituted for G50F for hydrophilic ligands, which do not partitioninto detergent micelles. This gives a more rapid separation. Therecovery of specifically bound ligand is calculated in absolute terms:

bound ligand=(dpm (total)−dpm (non-spec.)×5/(2220×spec. act) pmol/ml

[1228] An aliquot of the pellets is resuspended and counted to calculaterecovery of unsolubilized receptors. The concentration of protein in thesolubilized supernatant is measured, for example, by measuring UVabsorbance at 280 nm against a detergent-buffer blank. (If necessary,the supernatant is diluted to get the absorbance on scale.) Proteinconcentration in the solution is approximately equal to the absorbanceat 280 nm.

[1229] Alternatively, the Lowry method is used. The above steps arerepeated without first prelabeling the receptors in the membrane.Instead, the solubilized supernatantis incubated in the absence andpresence of labeled ligand. Again, concentration of the labeled ligandis used that saturates the binding sites. Incubation is carried out for2 h at 4° C., and the binding is assayed by gel filtration as above. Thepellet is resuspended and assayed for residual binding to check overallrecovery. The molecular size of the receptors in solubilizedpreparations is estimated by a combination of gel filtrationchromatography and sucrose density gradient centrifugation in H2O andD2O. Affinity chromatography is the principal method use forpurification of all of the receptors, combined with gel permeation HPLC,and ion exchange. SDS PAGE is carried out on the final product. Affinitychromatography is carried out using immobilized SEB, and the column iseluted with acid buffer or different concentrations and ionic strengthsof eluting buffer.

[1230] Determination of amino-acid and oligonucleotide sequences of SAgreceptors

[1231] Receptor material is eluted from the SDS-PAGE, and the N-terminalamino acid sequence is determined. When free amino termini are notavailable, the purified receptor material must be subjected to partialhydrolysis. The specific cleavage of peptide bonds is performed withendoproteases, such as V8 protease or trypsin, or with chemicals such ascyanogen bromide(CNBR). The resulting peptides are separated by SDS-PAGEwhen they are over residues or by reverse phase HPLC. The peptides thusanalyzed are subjected to amino-acid sequence analysis with a gas phaseor solid phase sequencer.

[1232] Antibodies are raised against the peptides, and the resultantantibodies used to confirm that the peptide is a part of the receptor byimmunoprecipitation or Western blot.

[1233] To determine the full sequence of the receptor gene,oligodeoxynucleotide probes synthesized on the basis of peptidesequences are used to screen an appropriate cDNA library. Either amixture of relatively short oligonucleotides with all possible sequencesor a relatively short oligonucleotides with a sequence based on codonusage frequency is used. Genomic libraries as well as cDNA libraries arescreened to obtain genes for receptors and to deduce their amino acidsequence. The amino acid sequence deduced from the nucleotide sequenceis compared to the known sequences of other receptors. Among the usefulstructural information derived from the sequence analysis is thehydropathy profile. The presence of hydrophobic domains with a length ofapproximately 20 amino acids residues suggests that the regions aretransmembrane segments. Genomic or cDNA clones ligated into expressionvectors are used to transform suitable cell lines.

[1234] Alternatively, mRNA transcribed from these clones is injectedinto recipient cells such as Xenopus oocytes. The expression ofreceptors in these cells is confirmed by measuring ligand binding,reactivity of cell homogenates or membrane preparations with antibodiesor the responses induced by receptor agonists in recipient cells. Thedirect function of the receptors is elucidated by reconstitutingpurified receptors in phospholipid vesicles with or without othercomponents.

[1235] An additional method is based on the isolation of cDNA or genomicclones for receptors without using purified receptors. The structure ofreceptors and cellular responses to them is examined using these clones.Substantial amounts of receptor material is produced from these clones.Monoclonal antibodies to the SAg receptors are used to screen clones forreceptors derived from cDNA libraries constructed with expressionvectors.

[1236] Transfection of SAg receptor involves the ligation of thereceptor gene into an appropriate expression vector, transformation of asuitable bacterial host, and isolation of an individual bacterial colonycontaining the plasmid vector. The plasmid DNA is harvested from thelysed bacteria. The preferred method of purification of plasmid DNA foruse in transfections involves Triton-lysozyme equilibrium gradient. Thecells to be used in transfection are maintained in the log phase ofgrowth at all times. The calcium phosphate method is useful andefficient means for introduction of cloned genes in plasmid vectors intomammalian cells as described earlier in this document is preferred.However, the other methods given are useful as well. A partial list ofplasmid vectors and promoters suitable for transfection of culturedmammalian cell is given in Fraser, C. M., Expression of Receptor Genesin Cultured Cells in Receptor Biochemistry, A Practical Approach, Hulme,E. C., ed., Oxford University Press, pp. 263-275, 1993.

EXAMPLE 13 Avoiding Interference with SAg-Specific Antibodies

[1237] Naturally antibodies are found in mammals that are specific forthe SAg molecule (e.g., a Staph enterotoxin). Such antibodies bind andinterfere with the SAg expressed and secreted by transfected cells. Suchantibodies also hinder therapeutic action of SAg infused directly (asnative protein, peptide or fusion protein).

[1238] It is desirable to neutralize or otherwise remove such before thecells of this invention are administered to a subject. One way toachieve this is to pre-treat the subject with antiidiotypic antibodiesspecific for the variable region of SAg-specific antibodies. Another wayis to infuse SAg peptides that represent the major immunogenic portionsof the overall protein. Alternatively, SAg is immobilized to a solidsupport by covalent bonding and the blood or plasma is perfusedextracorporeally through a device containing the immobilized protein,thereby removing the antibodies by immunoadsorption. In anotherapproach, SAg-expressing cells (prokaryotic or eukaryotic) preferably ofhost origin, or phage displays, are encapsulated and used asimmunoadsorbents to binds circulating SAg-specific antibodies. Anorganoid containing these adsorbing cells is positioned subcutaneouslyor placed into the circulation via catheter and then removed once theadsorption process is complete. Alginate encapsulated cells expressingSAg are preferred but other known modes of cell encapsulation may beused. Liposomes with surface-bound SAg are another form ofimmunoabsorbent that are employed either as an organoid or by directinjection.

[1239] Induction of Immunological Tolerance

[1240] The induction of tolerance to epitopes of the SAg molecule whichinduce a humoral antibody response would be desirable. The portion ofthe SEA molecule which binds to natural antibodies is the linearsequence of residues 232-262. Immune tolerance is induced using thissequence by the method of Dintzis et al., Proc. Natl. Acad. Sci. 89:1113-1117 (1992). in which low molecular weight peptide arrays areadministered to patients with circulating antibodies to enterotoxins.The peptides are delivered parenterally or orally once weekly in dosesof 1-500 mg/kg for three to six weeks after which there is a reductionand disappearance of circulating antibody specific for the tolerogen.

[1241] After one or more of the foregoing treatments, native SAg or SAgconjugated to a monoclonal tumor-specific antibody and administered tothe host can now localize to tumor sites without diversion bycirculating SAg-specific antibodies.

[1242] Phage Displayed SAgs

[1243] Phage display technology may also be used neutralize circulatinganti-enterotoxin antibodies. The SAg and/or SAg receptor is expressed atthe surface of bacteriophage as a fusion protein with the gene VIIIprotein (gVIIIp). This phage-displayed SAg fusion protein retains theproperties of the natural protein. For this invention, the filamentousphage vector f88-4 which forms a fusion protein between the C terminusof the inserted gene product and the N terminus of gVIIIp is used. Thephage expressing SEA is injected intravenously into patients that havenatural antibodies to SEA. The amount of phage (transducing units)required to neutralize the circulating pool of antibodies ispredetermined by antigen binding inhibition assay. The number oftransducing units required to neutralize the pool of circulating SEAspecific antibodies is administered intravenously. Shortly after thisinjection, the host is ready for treatment with active SEA which is nolonger hindered from finding its “target,” i.e., enterotoxin receptorsexpressed by tumor cells or T cells.

[1244] SEA clone pKH-X35 is employed. PCR with Vent Polymerase 9NEB isused to mutate the 5′- and 3′-ends of the SEA gene for cloning intof88-4. The construct is as follows. The 5′ oligonucleotide used is5′-CTCCAAGCTTTGVCCAGCGAGAAAAGCGAAG-3′. Two 3′ oligonucleotide primersare used. For the construct with the five amino acid linker between SEAand gVIIIp (SEA L), the primer5′-GCCTCCTGCAGATCCACCGCCTCCGGATGT-ATATAAATATATATC-3′ and for thenon-linker version (SEA-P); 5′-GCCTCCTGCAGATGTATATAAATATATATC-3′ areused. The two SEA PCR products are cut with HindIII and PstI and clonedinto f88-4. They are transformed by electroporation into E. coli strainDH5a and sequenced. Phage are produced by growing the transformedbacteria overnight in 0.5 L of broth with 20 mg/ml tetracycline. Theculture is pelleted twice (800×g for 15 min) and the phage precipitatedout of the cleared supernatant by the addition of 0.15 vols. of PEG/NaClsolution (17% PEG 8000, 19% NaCl in water). After incubation at 4° C.for 2 hours, the phage are resuspended in TBS and sterile-filteredthrough a 0.22-m membrane. Phage are selected by the micropanningtechnique and by cell binding. Binding to antibody is assessed byattaching mAb to the surface of 96-well ELISA plates, blocking with 1%BSA, incubating with 100 mg/ml of SEA or PBS as a control and thenincubating with the various phage preparations for >2 hours at 4° C. Thephage is then eluted with 0.1 M HCl pH 2 (adjusted with glycine) for 10minutes, neutralized and used to infect starved E. coli MC0161 F′ Kan.The infected bacteria are then spread on tetracycline (20 mg/ml) LB agarplates. After overnight culture tetracycline resistant colonies arecounted representing the number of transducing units (TU) recovered. Todetermine the number of SEA-bearing phage among thetetracycline-resistant colonies, colony blotting is performed bystandard techniques probing with a ³²P-labeled SEA probe. An antibodybased variant of this technique is involves probing with a rabbitanti-SEA serum as for Western blots.

[1245] Chimeric Enterotoxins

[1246] Likewise, hybrid or chimeric SAgs that are non-immunogenic areused to stimulate cells. When these molecules are injected into hoststhat have natural antibodies, they are not rapidly eliminated from thecirculation. Such chimeric molecules lacking the binding site fornatural antibodies preserve the T cell mitogenic and cytokine-inducingproperties of the native SAg. A peptide sequence from another SAg towhich antibodies do not exist is substituted using genetic orbiochemical methods well known in the art. This is particularly usefulin the case of enterotoxins such as SEB or SEA to which a largepercentage of humans have naturally occurring circulating antibodies.The antibody binding region of these molecules near the C terminalregions is delineated. The substitution of the antibody bindingsequences in SEA or SEB for sequences from SEE or SED to which a verysmall number of humans have circulating antibodies markedly enhances thetumor killing efficacy of the injected chimeric enterotoxins.

[1247] A hybrid molecule consisting of a 26 amino acid peptidecorresponding to the N-terminal portion of SEA, the loop structure ofSEA, a conserved mid-molecular sequence of SEA and SEB, and a C terminalsequence of SEB was synthesized in collaboration with Multi-PeptideSystems, La Jolla, Calif. Peptides were prepared using a variation ofMerrifield's original solid phase procedure in conjunction withsimultaneous multiple peptide synthesis using t-Boc chemistries.Peptides were cleaved from the resins using simultaneous liquid HFcleavage. The cleared peptides were then extracted with acetic acid andethyl ether and lyophilized. Reverse phase HPLC analysis and massspectral analysis revealed a single major peak with the molecular weightcorresponding closely to theoretical.

[1248] Synthetic SAgs

[1249] Amino acid sequences of SEA and SEB known to be involved in theinteraction with the TCR and MHC class II molecules are retained. Theloop structure of SEA is retained because it is devoid of histidinemoieties that are associated with the emetic response. Residues 1-10 ofthe N-terminal region of SEA are retained because they have MHC class IIbinding activity. The loop structure of SEA is retained because it andassociated disulfide linkages are considered to be important for Tlymphocyte mitogenicity, stabilization of the molecule, and resistanceto in vivo degradation. A conservd sequence in the central portion ofSEA and SEB adjacent to the disulfide loop (amino acids 107-114) wasretained. Histidine moieties are deleted from the molecule because oftheir association with the emetic response.

[1250] Synthesis Procedure

[1251] The preparation of all pptides was carried out using a variationof Merrifield's original solid phase procedures in conjunctio with themethod of Simultaneous Multiple Peptide Synthesis using t-Bocchemistries (Merrifield R B I, J. Amer. Chem. Soc. 85:2149-2154 (1963));Houghten R A, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985); andHoughten R A et al., Intact. J. Peptide Protein Res. 27:673-678 (1985)).

[1252] 4-methylbenzhydrylamine (mBHA) and phenylacetamidomethyl (PAM)resins were purchased from Advanced Chemtech (Louisville, Ky.) andBachem (Torrance, Calif.), respectively. All of the amino acidscontained the t-butyloxycarbonyl (t-Boc)-amino protecting group and werepurchased from Bachem. The side chain protecting groups included benzyl(threonine, serine and glutamic acid), chlorobenzyloxycarbonyl (lysine),bromobenzyloxycarbonyl (tyrosine), cyclohexyl (aspartic acid), p-toluenesulfonyl (arginine), formyl (tryptophan), methyl benzyl (cysteine), anddinitrophenyl or benzyloxycarbonyl (histidine). Cysteine with the HFstable acetamidomethyl (ACM) protecting group was used, upon request,for internal cysteines. Each lot of amino acid derivative was tested bymelting point analysis. Reagent grade methylene chloride (CH2C12),isopropanol (IPA), and dimethylformamide (DMF) were obtained from FisherScientific (Tustin, Calif.). diisopropylcarbodiimide (DIPCDI) anddiisopropylethylamine (DIEA) were purchased from Chem Impex (Wood Dale,Ill.). Trifluoroacetic acid was purchased from Halocarbon (Hackensack,N.J.).

[1253] The appropriate resin, mBHA for C-terminal amides and PAM forC-terminal acids, was weighed with a Mettler AE 240 balance (Highstown,N.J.) into separate polypropylene mesh (74 mm) packets which had beenpre-sealed on 3 of 4 sides using a TSW TISH-300 Impulse Sealer (SanDiego Bag and Supply; San Diego, Calif.). Each packet was alsopre-labeled with a reference code using a KOH I NOOR Rapidograph penwith graphite based ink to allow them to be easily identified duringresin addition and during the synthesis process. Each packet was thencarefully sealed completely to make sure there would be no resinleakage. All the resin containing packets (up to 150) were then placedin a common Nalgene bottle. Enough CH2C12 to cover all the packets wasthen added to the bottle, which was then capped and vigorously shakenfor 30 seconds on an Eberbach Shaker (Fisher Scientific; Tustin, Calif.)to wash and swell the resin. The CH2C12 solution was then removed. Allsubsequent steps involved the addition of enough solvent to cover allthe packets and vigorous shaking to ensure adequate solvent transfer.The N—t-Boc was removed by acidolysis using a solution of 55% TFA inCH2C12 for 30 minutes, leaving the TFA salt of the a-amino group. TheTFA wash solution was then removed. The packets were then washed for 1min with CH2C12 (2×), IPA (2×) and CH2C12 (2×) to squeeze out excess TFAand to prepare for neutralization. The TFA salt was neutralized bywashing the packets three times with 5% DIEA in CH2C12 for two minuteseach. This was followed by two washes with CH2C12 to remove excess base.

[1254] The resin packets were then removed from the common Nalgenebottle and sorted according to computer generated checklists inpreparation for coupling. This was double checked to ensure the packetswere added to the correct amino acid solution. The packets were thenadded to bottles containing the appropriate 0.2 M amino acid in CH2C12and/or DMF depending on solubility. These solutions were also preparedusing computer generated information. An equal volume of 0.2 M DIPCDIwas then added to activate the coupling reaction. The bottles were thenshaken for one hour to ensure complete coupling. At completion, thereaction solution was discarded and the packets were washed. with DMFfor 1 min to remove excess amino acid and the by-product,diisopropylurea. A final CH2C12 wash as then used to remove DMF. Thepackets were then removed from their individual coupling bottles andplaced back into the common Nalgene bottle.

[1255] The peptides were then completed by repeating the same procedurewhile substituting for the appropriate amino acid at the couplingjuncture. The packets were then taken through a final acidolysis alongwith subsequent CH2C12, IPA and CH2C12 washes to leave the peptides inthe TFA salt form. The packets were then dried in preparation for thenext process.

[1256] Final side chain deprotection and cleavage of the anchoredpeptide from the resin was achieved through simultaneous liquid HFcleavage (Houghten R A et al., supra.

[1257] Gaseous N₂, HF, and argon were acquired from Air Products (SanDiego, Calif.). Anisole was purchased from Aldrich Chemical Co.(Milwaukee, Wis.). Acetic acid (HOAc) and ethyl ether were purchasedfrom Fisher Scientific (Tustin, Calif.). Each packet along with a Tefloncoated stir bar was placed into an individual reaction vessel of amulti-vessel hydrogen fluoride apparatus (Multiple Peptide Systems; SanDiego, Calif.). An amount of anisole equaling 7.5% of the expectedvolume of HF was then added to act as a carbonium ion scavenger. Thereaction tubes were lubricated with vacuum grease at the point whereeach contacts the apparatus and sealed onto the HF system. The systemwas then purged with N₂ while cooling the reaction vessels to −70° C.using an acetone/dry ice bath. HF (g) was condensed to the desired leveland temperature elevated to −10° C. using ice and water. The reactionwas allowed to proceed for 90 minutes with the temperature slowly risingfrom −10° C. to 0° C. HF was removed using a strong flow of N₂ for 90minutes followed by the use of aspirator vacuum for 60 minutes whilemaintaining the temperature at 0° C. The reaction vessels were thenremoved from the apparatus and capped. The residual anisole was removedwith two ethyl ether washes. The peptide was then extracted with two 10%HOAc washes. A 50 ml sample of the crude peptide was taken and run on ananalytical Beckman 338 Gradient HPLC System (Palo Alto, Calif.) using aVydac C18 column to profile the initial purity of the compound. Thecrude peptide was then lyophilized twice on a Virtis Freezemobile 24Lyophilizer, weighed and stored under argon.

[1258] Analytical RP-HPLC was used to determine the homogeneity andapproximate elution conditions of the peptides produced. HPLC gradeacetonitrile (ACN) was purchased from Fisher Scientific (Tustin,Calif.). HPLC grade TFA was obtained from Pierce Chemicals (Rockford,Ill.). RP-HPLC analysis was carried out on a Beckman 338 Gradient HPLCsystem (Palo Alto, Calif.) equipped with a BioRad AS-100 autosampler anda Shimadzu CR4A integrator. The column used for all analyses thisquarter was a Vydac C-18 column (4.6×250 mm). The solvent system usedwas 0.05% aqueous TFA (A) and 0.05% TFA in ACN (B) with a flow rate of 1ml/min. Absorbance was measured at 215 nm. Most peptides were analyzedusing the following special gradient; 5.60% (B) in 28 minutes.Hydrophobic peptides were analyzed using the following special gradient:5-40% (B) in 9 minutes, 40-90% (B) for 10 additional minutes, 95% (B)for the last 9 minutes.

[1259] Analytical data was reviewed. The product peak was identified andmarked based upon knowledge of common impurities and the use ofpredicted HPLC retention times.

[1260] Peptides that did not meet normal purity requirements for crudematerial were purified using preparative RP-HPLC techniques. HPLC gradeacetonitrile (CAN) was purchased from Fisher Scientific (Tustin,Calif.). HPLC grade TFA was obtained from Pierce Chemicals (Rockford,Ill.). Purification was carried out on a Waters Delta Prep 3000 with aPreparative Waters Prep Pak Module Radial Compression C18 column (5cm×25 cm, 10-20 m). The solvent system used was 0.05% aqueous TFA (A)and 0.05% TFA in ACN (B). The crude peptides were solubilized in anHOAc/H20 mixture and injected onto the column with 0.25% to 0.50% ACNper minute linear gradient. The absorbance was measured at 230 nm and 40ml fractions were collected upon elution with an ISO Fraction Collector(Lincoln, Nebr.). The preparative profile was reviewed and selectedfractions were analyzed by analytical RP-HPLC. The analytical data wasreviewed and fractions were combined and lyophilized. The lyophilizedmaterial was weighed, sampled for a final analytical RP-HPLC analysisand stored under argon in powder form. This process was repeated if thepurity level attained was not sufficient.

[1261] Mass spectral analysis was used to determine the molecular weightof the peptides produced. 95% ethanol was purchased from FisherScientific (Tustin, Calif.). HPLC grade TFA was obtained from PierceChemicals (Rockford, Ill.). Nitrocellulose matrices (targets) werepurchased from Applied Biosystems (Foster City, Calif.).

[1262] The samples were solubilized in a 1:1 solution of 95% ethanol and0.1% TFA (aqueous). The samples were applied to a nitrocellulose matrix(Target). The mass spectra were obtained using an ABI Bio-Ion 20 MassSpectrometer (Foster City, Calif.). The apparatus makes use of plasma;desorption ionization via a Cf252 source. The ionized molecules are thenanalyzed via time-of flight. An accelerating voltage of 15,000 V is usedto accelerate the particles.

[1263] The Protocol for Intramolecular Disulfide Bridge:

[1264] Dissolve crude peptide (300-500 mg) in 200 ml of deoxygenatedwater and adjust the pH to 8.5 using NH4OH 28%=Solution A. Note: If thepeptide is not very soluble in water, some MeOH can be added.

[1265] Dissolve 0.5 g K3Fe(CN)6 in 200 ml of deoxygenated water andadjust the pH to 8.5 using NH4OH 28%=Solution B. Note: 0.5 g K3Fe(CN)6is an average value for 500 mg of a 10 mer peptide. The excess ofK3Fe(CN)6 should be approximately 3×. It can be adjusted.

[1266] Solution A is then dropped slowly into solution B over a 2 hourperiod. The mixture is then allowed to react, for an additional 1 hourwith stirring. The pH is then adjusted to 4.0-4.5 with 10% ACTH. Thissolution is injected directly into a preparative RP-HPLC. The major peakis then collected. This “pseudo dilution” technique favors theintramolecular disulfide. Therefore, the major peak is the cyclicproduct.

[1267] The chimeric enterotoxin molecule was tested in normal rabbitsand rabbits with established VX2 carcinoma. It was administeredintravenously and peripherally with adjuvant. The chimeric molecule (1mg/ml) was diluted initially in 1 ml of sterile H20. When the solutionwas clear, 9 ml of normal saline was added. The solution was filteredthrough a 0.45 m filter and stored in 0.5-1 ml aliquots. Dosage rangedfrom 2.6-5.0 mg/kg and was described over 3 minutes via the lateral earvein in a volume of 0.05 ml diluted further in 1.0 ml of 0.15 M NaCl:

[1268] The i.v. line was then washed with 3 ml of 0.15M NaCl.

[1269] In two animals, the temperature rose only 0.3 F over the ensuing24 hours and there was no discernible toxicity over the ensuing 14 daysof observation. One animal was described a second dose of the chimericmolecule in pluronic acid triblock adjuvant. This was described in adose of 8.5 mg subcutaneously in each thigh with a total dose 5 mg/kg.The pluronic acid triblock preparation was prepared as follows: 4.23 ccPBS; 0.017 cc Tween; 0.05 cc Squalene; and 0.25 cc Pluronic. The PBS andTween were mixed first then squalene was added followed by pluronicacid. The total mixture was vortexed for 3-4 minutes. Two ml of aboveplus 0.34 ml of the chimeric protein (34 mg) plus 1.66 cc PBS were addedto the mixture. The mixture was vortexed vigorously for 1-2 minutes. Oneml was injected into each thigh (total vol. injected was 0.17 ml or 17mg protein or 5 mg/kg).

[1270] For nearly 5 weeks after injection, no adverse effects werenoted. The tumor showed slow, but progressive growth over this period oftime. To date, the chimeric enterotoxin molecule appears to be safe inanimals and no untoward side effects were demonstrated. The adjuvantused for these studies was the pluronic acid triblock copolymer whichhas been used to boost the immune response to various antigens in animalmodels and which is under testing at this point in humans with hepatitisand herpes simplex infections. Other adjuvants including those preparedin water and oil emulsion and aluminum hydroxide to administer variousSAgs in vivo to tumor bearing rabbits were also used.

[1271] Additionally, enterotoxins such as SEE, SED, SEC, and TSST-1 areused to prepare hybrid molecules containing amino acid sequences andhomologous to the enterotoxin: family of molecules. To this extent,mammary tumor virus sequences, heat shock proteins, stress peptides.Mycoplasma and mycobacterial antigens, and minor lymphocyte stimulatingloci bearing tumoricidal structural homology to the enterotoxin familyare useful as anti-tumor agents. Hybrid enterotoxins and other sequenceshomologous to the native enterotoxins are immobilized or polymerizedgenetically or biochemically to produce the repeating units andstoichiometry required for (a) binding of accessory cells to Tlymphocytes and (b) activation of T lymphocytes.

EXAMPLE 14 Pharmaceutical Compositions and their Manufacture

[1272] The pharmaceutical compositions may be in the form of alyophilized particulate material, a sterile or aseptically producedsolution, a tablet, an ampoule, etc. Vehicles such as water (preferablybuffered to a physiological pH such as PBS or other inert solid orliquid material may be present. In general, the compositions areprepared by being mixed with or dissolved in, bound to or otherwisecombined with one of more water-insoluble or water-soluble aqueous ornon aqueous vehicles, if necessary together with suitable additives andadjuvants. It is imperative that the vehicles and conditions shall notadversely affect the activity of the conjugate. Water as such iscomprised within the expression vehicles.

[1273] A suitable therapeutic composition is used in the treatment ofcancer of any kind including but not limited to carcinomas, sarcomas,lymphomas, leukemias and comprises a combination of:

[1274] (1) a recombinant DNA molecule encoding SAg in combination with,preferably fused with, another recombinant DNA sequence encoding anotherprotein;

[1275] (2) a recombinant DNA molecule encoding SAg-in combination withanother peptide or polypeptide; or

[1276] (3) a recombinant DNA molecule encoding a protein other than aSAg in combination with a SAg peptide or polypeptide.

[1277] These compositions that may comprise more than one components areadministered together or sequentially and they may be combined(separately or together) with a delivery vehicle, preferably liposomesas disclosed herein.

[1278] Upon entering its intended or targeted cells, the therapeuticcomposition leads to the production of SAg and a second protein that mayresult in (a) apoptosis of the cancer cell and (b) with or without suchapoptosis, the activation of effector cells of the immune system,including any or all of the following: cytotoxic T cells, NKT cells, NKcells, T helper cells and macrophages. The present therapeuticcompositions are useful for the treatment of cancers, both primarytumors and tumor metastases.

[1279] Use of the present therapeutic composition overcomes thedisadvantages of traditional treatments for metastatic cancer. Forexample. compositions of the present invention can target dispersedmetastatic cancer cells that cannot be treated using surgery. Inaddition, administration of such compositions is not accompanied by theharmful side effects of conventional chemotherapy and radiotherapy.

[1280] A therapeutic composition also comprises a pharmaceuticallyacceptable carrier defined as any substance suitable as a vehicle fordelivering a nucleic acid molecule (alone or in some combination with aprotein) to a suitable in vivo or in vitro site. Preferred carriers arecapable of maintaining DNA in a form that is capable of entering thetarget cell and being expressed by the cell.

[1281] Preferred carriers include: (1) those that transport, but do notspecifically target a nucleic acid molecule to a cell (referred toherein as “non-targeting carriers”); and (2) those that deliver anucleic acid molecule to a specific site in an animal or a specific cell(“targeting carriers”). Examples of non-targeting carriers are water,phosphate buffered saline (PBS), Ringer's solution, dextrose solution,serum-containing solutions, Hank's balanced salt solution, otheraqueous, physiologically balanced solutions, oils, esters and glycols.Aqueous carriers can contain suitable additional substances whichenhance chemical stability and isotonicity, such as sodium acetate,sodium chloride, sodium lactate, potassium chloride, calcium chloride,and other substances used to produce phosphate buffer, Tris buffer, andbicarbonate buffer and preservatives, such as thimerosal, m- ando-cresol, formalin and benzyl alcohol.

[1282] Preferred substances for aerosol delivery include surfactantsubstances such as esters or partial esters of fatty acids containingfrom about 6-22 carbon atoms. Examples are esters of caproic, octanoic,lauric, palmitic, stearic, linoleic, linolenic, olesteric, and oleicacids.

[1283] Other carriers can include metal particles (e.g., colloidal goldparticles) for use with, for example, a biolistic gun through the skin.

[1284] Therapeutic compositions of the present invention can besterilized by conventional methods and may be lyophilized.

[1285] The compositions of the present invention are delivered using adelivery vehicle that can be modified to target a particular site in asubject. Suitable targeting agents include ligands capable ofselectively (i.e., specifically) binding to another molecule at aparticular site. Examples are antibodies, antigens, receptors andreceptor ligands. For example, an antibody specific for an antigen onthe surface of a cancer cell can be placed on the outer surface of aliposome delivery vehicle to target the liposome to the cancer cell. Bymanipulating the chemical formulation of the lipid portion of a liposomepreparation, it is possible to modulate its extracellular orintracellular targeting. For example, the charge of the lipid bilayer ofa liposome surface can be varied chemically to promote fusion with cellshaving particular charge characteristics. Preferred liposomes comprise acompound that targets the liposome to a tumor cell, such as a ligand onthe outer surface of the liposome that binds a molecule on the tumorcell surface.

[1286] Although the DNA constructs of the present invention can beadministered in naked form, a liposome is a preferred vehicle fordelivery in vivo. A liposome can remain stable in an animal for asufficient amount of time, at least about 30 minutes, more preferablyfor at least about 1 hour and even more preferably for at least about 24hours, to deliver a nucleic acid molecule to a desired site.

[1287] A liposome of the present invention comprises a lipid compositionthat can fuse with the plasma membrane of the targeted cell to deliverthe encapsulated nucleic acid molecule into a cell. Preferably, theliposomes' transfection efficiency is about 0.5 mg DNA per 16 nmol ofliposome delivered to about 10⁶ cells, more preferably about 1.0 mg DNAper 16 nmol of liposome delivered to about 10⁶ cells, and even morepreferably about 2.0 mg DNA per 16 nmol of liposome delivered to about10⁶ cells.

[1288] For use in the present invention, any liposome that is used inart-recognized gene delivery methods is appropriate. Preferred liposomeshave a polycationic lipid composition and/or a cholesterol backboneconjugated to polyethylene glycol.

[1289] Complexing a liposome with nucleic acids for uses describedherein is achieved using conventional methods. A suitable concentrationof DNA to be added to a liposome preparation a concentration that iseffective for delivering a sufficient amount of DNA molecules to a cellso that the cell can produce sufficient SAg and/or a other transducedprotein to induce tumoricidal activity or to stimulate or regulateeffector cells in a desired manner. Preferably, between about 0.1 mg and10 mg of DNA is combined with about 8 nmol liposomes; more preferably,between about 0.5mg and 5 mg of DNA is used even more preferably, about1.0 mg of DNA is combined with about 8 nmol liposomes.

[1290] Another preferred delivery system is the sickled erythrocytecontaining the nucleic acids of choice a given in Example 6. The sicklederythorcytes undergo ABO and RH phenotyping to select compatible cellsfor delivery. The cells are delivered intravenously or intrarterially ina blood vessel perfusing a specific tumor site or organ e.g. carotidartery, portal vein, femoral artery etc. over the same amount of timerequired for the infusion of a conventional blood transfusion. Thequantity of cells to be administered in any one treatment would rangefrom one tenth to one half of a full unit of blood. The treatments aregenerally given every three days for a total of twelve treaments.However, the treatment schedule is flexible and may be given for alonger of shorter duration depending upon the patients response.

[1291] Another preferred delivery vehicle is a recombinant virusparticle, for example, in the form of a vaccine. A recombinant virusvaccine of the present invention includes: the DNA encoding thetherapeutic composition packaged in a viral coat that allows entrance ofthe transducing DNA into a cell and its expression. A number ofrecombinant virus particles can be used, for example, alphaviruses,poxviruses, adenoviruses, herpesviruses, arena virus and retroviruses.

[1292] Also useful as a delivery vehicle is a “recombinant cellvaccine,” preferably tumor vaccines, in which allogeneic (thoughhistocompatible) or autologous tumor cells are transfected with a DNApreparation encoding the therapeutic proteins or peptides to beexpressed. The cells are preferably irradiated and then administered toa patient by any of a number of known injection routes.

[1293] The therapeutic compositions that are administered by “tumor cellvaccine,” includes the recombinant molecules without carrier. Treatmentwith tumor cell vaccines is useful for primary or localized tumors aswell as metastases. When used to treat metastatic cancer, which includesprevention of further metastatic disease, as well as, the cure existingmetastatic disease.

[1294] As used herein, the term “treating” a disease includesalleviating the disease or any of its symptoms and/or preventing thedevelopment of a secondary disease resulting from the occurrence of theinitial disease.

[1295] An “effective treatment protocol” includes a suitable andeffective dose of an agent being administered to a subject, given by asuitable route and mode of administration to achieve its intended effectin treating a disease.

[1296] Effective doses and modes of administration for a given diseasecan be determined by conventional methods and include, for example,determining survival rates, side effects (i.e., toxicity) andqualitative or quantitative, objective or subjective, evaluation ofdisease progression or regression. In particular, the effectiveness of adose regimen and mode of administration of a therapeutic composition ofthe present invention to treat cancer can be determined by assessingresponse rates. A “response rate” is defined as the percentage oftreated subjects that responds with either partial or completeremission. Remission can be determined by, for example, measuring tumorsize or by microscopic examination of a tissue sample for the presenceof cancer cells.

[1297] In the treatment of cancer, a suitable single dose can varydepending upon the specific type of cancer and whether the cancer is aprimary tumor or a metastatic form. One of skill in the art can testdoses of a therapeutic composition suitable for direct injection todetermine appropriate single doses for systemic administration, takinginto account the usual subject parameters such as size and weight. Aneffective anti-tumor single dose of a therapeutic recombinant DNAmolecule or combination thereof is an amount sufficient amount to resultin reduction, and preferably elimination, of the tumor after the DNAmolecule or combination has transfected cells at or near the tumor site.

[1298] A preferred single dose of SAg-encoding DNA molecule or fusionproduct thereof is an amount that, when transfected into a target cellpopulation, leads to the production of SAg in an amount, per transfectedcell, ranging from about 250 fentograms (fg) to about 1 mg, preferablyfrom about 500 fg to about 500 pg and more preferably from about 1 pg toabout 100 pg.

[1299] When the SAg-encoding DNA is combined with a second DNA moleculeencoding a second protein product, an effective single dose of a thesecond DNA molecule is an amount that when transfected into a targetcell population leads to the production of the second protein product inan amount, per transfected cell, ranging from about 10 fg to about 1 ng,more preferably from about 100 fg to about 750 pg.

[1300] An effective cancer-treating single dose of SAg-encoding DNA anda second DNA molecule encoding a second protein when administered to asubject using a non-targeting carrier, is an amount capable of reducing,and preferably eliminating, the primary or metastatic tumor followingtransfection by the recombinant molecules of cells at or near the tumorsite. A preferred single dose of such a therapeutic composition is fromabout 100 mg to about 4 mg of total recombinant DNA, more preferablyfrom about 200 mg to about 2 mg, most preferably from about 200 mg toabout 800 mg of total recombinant molecules.

[1301] A preferred single dose of liposome-complexed, SAg-encoding DNA,is from about 100 mg of total DNA per 800 nmol of liposome to about 4 mgof total DNA molecules per 32 mmol of liposome, more preferably fromabout 200 mg per 1.6 mmol of liposome to about 3 mg of total recombinantDNA per 24 mmol of liposome, and even more preferably from about 400 mgper 3.2 mmol of liposome to about 2 mg per 16 mmol of liposome.

[1302] One of skill in the art recognizes that the number of dosesrequired depends upon the extent of disease and the response of anindividual to treatment. Thus, according to this invention, an effectivenumber of doses includes any number required to cause regression ofprimary or metastatic disease.

[1303] A preferred treatment protocol comprises monthly administrationsof single doses (as described above) for up to about 1 year. Aneffective number of doses (per individual) of a SAg-encoding DNAmolecule and a second DNA molecule encoding a second protein, whenadministered in a non-targeting carrier or when complexed withliposomes, is from about 1 to about: 10 dosings, preferably from about 2to about 8 dosings, and even more preferably from about 3 to about 5dosings. Preferably, such dosings are administered about once every 2weeks until signs of remission appear, followed by about once a monthuntil the disease is gone.

[1304] The therapeutic compositions can be administered by any of avariety of modes and routes, including but not limited to, localadministration into a site in the subject animal, which site containsabnormal cells to be destroyed. An example is the local injection withinthe area of a tumor or a lesion. Another example is systemicadministration.

[1305] Therapeutic compositions that are best delivered by localadministration include recombinant DNA molecules

[1306] (a) in a non-targeting carrier (e.g., “naked” DNA molecules astaught in Wolff K et al., 1990, Science 247, 1465-1468); and

[1307] (b) complexed to a delivery vehicle.

[1308] Suitable delivery vehicles for local administration includeliposomes, and may further comprise ligands that target the vehicle to aparticular site.

[1309] A preferred mode of local administration is by direct injection.Direct injection techniques are particularly useful for injecting thecomposition into a cellular or tissue mass such as a tumor mass or agranuloma mass that has been induced by a pathogen. Thus, the presentrecombinant DNA molecule complexed with a delivery vehicle is preferablyinjected directly into, or locally in the area of, a tumor mass or asingle cancer cell.

[1310] The present composition may also be administered in or around asurgical wound. For example, a patient undergoes surgery to remove atumor. Upon removal of the tumor, the therapeutic composition is coatedon the surface of tissue inside the wound or injected into areas oftissue inside the wound. Such local administration will treat cancercells that were not successfully removed by the surgical procedure, aswell as prevent recurrence of the primary tumor or development of asecondary tumor in the surgical area.

[1311] Therapeutic compositions that are best delivered by systemicadministration include recombinant DNA molecules complexed to a tumorbinding ligand or a ligand that binds to the tumor vasculature orstroma. Examples are antibodies, antigens, receptor, receptor ligand ora targeted delivery vehicle as disclosed herein. These delivery vehiclesmay be liposomes into which are incorporated targeting ligands,preferably ligands that targeting the vehicle to the site of tumor cellsor another type of lesion. For cancer treatment, ligands thatselectively bind to cancer cells, or to cells within the area of acancer cell, are preferred. Systemic administration is used to treatprimary or localized tumors and, in particular, tumor metastases whereinthe cancer cells are dispersed. Systemic administration is advantageouswhen targeting cancer in organs, especially those difficult to reach fordirect injection, (e.g., heart, spleen, lung or liver).

[1312] Preferred modes and routes of systemic administration includeintravenous injection and aerosol, oral and percutaneous (topical)delivery. Intravenous injection methods and aerosol delivery areperformed conventionally. Oral delivery is achieved preferably bycomplexing the therapeutic composition to a carrier capable ofwithstanding degradation by digestive enzymes in the subject's digestivesystem. Examples of such carriers, includes plastic capsules or tabletsas are known in the art. For topical delivery, the therapeuticcomposition is mixed with a lipophilic reagent (e.g., DMSO) that canpass into the skin.

[1313] The therapeutic compositions and methods of the present inventionare intended for animals, preferably mammals and birds, in particularhouse pets, farm animals and zoo animals as these terms are generallyunderstood. By “farm animals” are intended animals that are eaten orthose that produce useful products (e.g., wool-producing sheep).Examples of preferred animal subjects to be treated are dogs, cats,sheep, cattle, horses and pigs. The present compositions and methods areeffective in inbred and outbred animal species. Most preferably, theanimal is a human.

[1314] Another component useful in combination with the therapeuticnucleic acids of this invention is an adjuvant suited for use with anucleic acid-based vaccine. Examples of adjuvant-containing compositionsinclude

[1315] 1) SAg-encoding DNA and a second DNA encoding a recombinantprotein; or

[1316] 2) SAg-encoding DNA combined with another peptide or polypeptide;or

[1317] 3) DNA encoding a second recombinant protein and a SAg peptide orpolypeptide.

[1318] As indicated above, effective doses of a SAg-encoding DNAcombined with a second DNA molecule, or a vaccine nucleic acid moleculeare determined conventionally by those skilled in the art. One measureof an effective dose is that produces a sufficient amount of SAg andsecond protein to stimulate effector cell immunity in a manner thatenhances the effectiveness of the vaccine. Adjuvants of the presentinvention are particularly suited for use in humans because manytraditional adjuvants (e.g., Freund's adjuvant and other bacterial cellwall components) are toxic whereas others are relatively ineffective(e.g., aluminum-based salts and calcium-based salts).

EXAMPLE 15 General Procedures for In Vivo and Ex Vivo Sensitization toProduce Tumor Specific Effector Cells for Adoptive Immunotherapy

[1319] After 9-12 days of tumor growth (approximately 8 mm in diameter),tumor-draining inguinal LN are removed sterilely. Lymphocyte suspensionsare prepared by teasing LN with needles followed by pressing with theblunt end of a 10-ml plastic syringe in HBSS. Tumor draining LN cellsare stimulated in vitro in a two-step procedure. Briefly, 4×10⁶ LN cellsin 2 ml of complete medium (CM) containing the SAg constructs areincubated in a well of 24-well plates at 37° C. in a 5% CO₂ atmospherefor 2 days. CM consisted of RPMI 1640 medium supplemented with 10%heat-inactivated FCS, 0.1 mM nonessential amino acids, 1 mM sodiumpyruvate, 2 mM freshly prepared L-glutamine. 100 mg/ml streptomycin, 100U/ml penicillin, a 50 mg/ml gentamycin, 0.55 mg/ml fungizone (all fromGIBCO, Grand Island, N.Y.) and 5×10⁻⁵M 2-mercaptoethanol (Sigma). Thecells were harvested, then washed and further cultured a 3×10⁵/well in 2ml of CM with IL-2. After 3-day incubation in IL-2, the cells arecollected and counted to determine the degree of proliferation. Finally,the cells are suspended in appropriate media for flow cytometricanalysis, evaluation of cytotoxicity and lymphokine secretion, or foradoptive immunotherapy.

EXAMPLE 16 General Adoptive Immunotherapy Protocol

[1320] Mice are injected with 2 to 3×10⁵ syngeneic tumor cells suspendedin 1 ml of HBSS to initiate pulmonary metastases. On day 3, activatedcells are given i.v. at numbers indicated generally 10⁶-10⁷. In someinstances, mice are also treated with 15,000 U IL2 in 0.5 ml HBSS twicedaily for 4 consecutive days to promote the in vivo function andsurvival of the activated cells. On day 20 or 21, all mice arerandomized, killed and metastatic tumor nodules on the surface of thelungs enumerated as previously described. If pulmonary metastasesexceeded 250, this number is arbitrarily assigned for statisticalanalysis. The significance of differences in metastases numbersbetween-experimental group is determined by the Wilcoxon rank sum test.Two sided p values of <0.1 are considered significant. Each experimentalgroup consists of at least five mice and no animal was excluded from thestatistical evaluation.

[1321] For testing SAg-glycosylceramide complexes and SAglipopolysaccharide complexes, additional models are used to assess thedependence of the antitumor effect on NKT cells. Natural killer T cells(NKT) lymphocytes express an invariant TCR encoded by the V14 and Ja281gene segments. Mice with a deletion of Ja281 exclusively lack V14. TheV14 NKT cell-deficient mice no longer mediate IL-12 induced rejection oftumors.

[1322] Also generated are transgenic mice lacking recombinationactivating gene(RAG) which preferentially generate V14 NKT cells butblock the development of other lymphocyte lineages, including NK, B, andT cells. These mice are termed V14 NKT mice. J281+/+(wild type), J281−/−(deleted of V14) and RAG−/−V14tgV8.2tg (deleted of NK, T and B cells butpreferentially generate V14 NKT cells) mice are injected

[1323] (a) with 2×10⁶ B16 or FBL-3 (erythroleukemia) cells in the spleento induce liver metastasis,

[1324] (b) intravenously with 3×10⁵ B16 or 2×10⁶ LLC (Lewis lungcarcinoma) cells for pulmonary metastases or

[1325] (c) subcutaneously with 2×10⁶ B16 cells (melanoma) forsubcutaneous tumor growth on day 0.

[1326] SAg conjugates or fusion proteins are injected in doses of 0.1 to50 mg on days 3, 5,7, and 9 after the day of tumor implantation. Controlanimals are injected with PBS on the same schedule. On day 14, the miceare killed and either metastatic nodules counted or GM3 melanomaantigens measured by radioimmunoassay as previously described. Forsubcutaneous tumor growth, injection of IL-12 or PBS is initiated on day5, and the mice are treated five times per week. The diameters of tumorsare measured daily with calipers. The sizes of the tumor are expressedas the products of the longest diameter times the shortest diameter (inmm²).

EXAMPLE 17 Preparation and Administration of DNA Liposome Complexes

[1327] A representative protocol for administration of DNA-liposomecomplexes is as follows: DNA liposome complexes are mixed immediatelyprior to injection by adding 0.1 ml of lactated Ringer's solution into asterile vial of plasmid DNA (20 mg/ml; 0.1 ml). An aliquot of thissolution (0.1 ml) is added at room temperature to 0.1 ml of 150 mM(dioleoylphosphatidylethanolamine/3β[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol) liposome in lactated Ringer's solution in a separatesterile vial. The DNA and liposome vials are prepared in accordance withFDA guidelines and quality control procedures. After incubation for 15minutes at room temperature, an additional 0.5 ml of sterile lactatedRinger's solution is added to the vial and mixed. The DNA liposomesolution (0.2 ml) is injected into the patient's tumor nodule understerile conditions at the bedside after administration of localanesthesia (1% lidocaine) using a 22-gauge needle. For catheterdelivery, the DNA liposome solution (0.6 ml) is delivered into theartery using percutaneous delivery. Additional protocols foradministration of DNA liposomal constructs are given in Nabel, G J,Methods for Liposome-Mediated Gene Transfer to Tumor Cells in vivo, in:Methods in Molecular Medicine, Gene Therapy Protocols, Robbins P ed.Humana Press, Totowa N.J. (1996). Cationic liposomes for delivery of DNAconstruct to the tumor endothelium are prepared by the method ofThurston et al., J. Clin Invest., 101: 1401-1413, (1998).

EXAMPLE 18 General Procedures for Administering Constructs in HumanTumor Models and Human Patients

[1328] The constructs described herein are tested for therapeuticefficacy in several well established rodent models which are consideredto be highly representative as described in “Protocols for ScreeningChemical Agents and Natural Products Against Animal Tumors and OtherBiological Systems (Third Edition)”, Cancer Chemother. Reports, Part 3,3: 1-112, which is hereby incorporated by reference in its entirety.Additional tumor models of carcinoma and sarcoma originating fromprimary sites and prepared as established tumors at primary and/ormetastatic sites are utilized to test further the efficacy of theconstructs.

EXAMPLE 19 General Procedures for Administering 1) siRNA(s) to T Cells,APCs & Tumor Cells, 2) Tumor Cells or Sickled Erythrocytes Transducedwith SAgs, 3) SAg-Activated T or NKT Cells in Human Tumor Models andHuman Patients

[1329] A. Tumor Cells Transduced with SAg Nucleic Acids alone orCotransfected with Oncogenes or Nucleic Acids Encoding Potent Immunogensand Bacterial Products

[1330] In a representative protocol, using the B16 melanoma or A20lymphoma or other models given above, 10⁵-10⁷ transfected tumor cellsare implanted subcutaneously and 1-6 months later 10⁵-10⁷ untransfectedtumor cells, are implanted. In the case of tumor cells cotransfectedwith several therapeutic nucleic acids, controls are establishedconsisting of groups transfected with only one of the nucleic acids.These single transfectants are administered on the same schedule as thecotransfectants and assessed for capacity to prevent or reverse tumorgrowth compared to positive controls receiving tumor alone. The animalsreceiving the siRNA or SAg transfected tumor cells show no-evidence orminimal of growth of the wild type tumor and prolonged survival comparedto the controls in which there is 100% appearance of the tumors. Thedifferences are statistically significant.

[1331] siRNA or SAg transfected tumor cells are also used to preventtumor outgrowth or treat established tumors as follows. Transfectedtumor cells, 10⁵-10⁷ are given at the time of tumor implantation or 3-10days after the appearance of established tumors. Results showstatistically significant arrest of tumor outgrown, tumor growth andprolongation of survival in treated animals compared to untreatedcontrols.

[1332] B. SAg Activated Effector T or NKT Cells & siRNA transfected APCs& T Cells

[1333] Effector T or NKT cells are generated as described elsewhere andare infused intravenously in doses of 10⁶-10⁸ into syngeneic hosts thathave pulmonary metastatic lesions established by injecting tumor cellsintravenously 3 to 12 days earlier. Twenty days later, the animals aresacrificed and pulmonary metastases measured in treated animals comparedto untreated controls. Results show statistically significant reductionin total number of pulmonary nodules and prolonged survival in thetreated group compared to untreated control.

EXAMPLE 20 General Test Evaluation Procedures for Constructs and SAgActivated Effector T or NKT Cells

[1334] Various SAg compositions described herein are tested fortherapeutic efficacy in several well established rodent models which areconsidered to be highly representative of a broad spectrum of humantumors. These approaches are described in detail in Geran, R. I. et al.,“Protocols for Screening Chemical Agents and Natural Products AgainstAnimal Tumors and Other Biological Systems (Third Edition)”, Canc.Chemother. Reports, Pt 3, 3:1-112, which is hereby incorporated byreference in its entirety.

[1335] A. Calculation of Mean Survival Time (MST)MST  (days)  is  calculated  according  to  the  formula:$\frac{S + {AS}_{({A - 1})} - {\left( {B + 1} \right){NT}}}{S_{({A - 1})} - {NT}}$

[1336] Day: Day on which deaths are no longer considered due to drugtoxicity. For example, with treatment starting on Day 1 for survivalsystems (such as L1210, P388, B16, 3LL, and W256): Day A=Day 6; DayB=Day beyond which control group survivors are considered “no-takes.”

[1337] S: If there are “no-takes” in the treated group, S is the sumfrom Day A through Day B. If there are no “no-takes” in the treatedgroup, S is the sum of daily survivors from Day A onward.

[1338] S(A-1): Number of survivors at the end of Day (A-1).

[1339] Example: for 3LE21, S(A-1)=number of survivors on Day 5.

[1340] NT: Number of “no-takes” according to the criteria given inProtocols 7.300 and 11.103.

[1341] B. T/C Computed for all Treated Groups

T/C=MST of treated group/MST of control group×100

[1342] Treated group animals surviving beyond Day Bare eliminated fromcalculations (as follows): No. of survivors in treated Percent of“no-takes” group beyond Day B in control group Conclusion 1 Any percent“no-take” 2 <10 drug inhibition ³10 “no-takes” ³3 <15 drug inhibitions³15 “no-takes”

[1343] Positive control compounds are not considered to have “no-takes”regardless of the number of “no-takes” in the control group. Thus, allsurvivors on Day B are used in the calculation of T/C for the positivecontrol. Surviving animals are evaluated and recorded on the day ofevaluation as “cures” or “no-takes.”

[1344] Calculation of Median Survival Time (MedST)

[1345] MedST is the median day of death for a test or control group. Ifdeaths are arranged in chronological order of occurrence (assigning tosurvivors, on the final day of observation, a “day of death” equal tothat day), the median day of death is a day selected so that one half ofthe animals died earlier and the other half died later or survived. Ifthe total number of animals is odd, the median day of death is the daythat the middle animal in the chronological arrangement died. If thetotal number of animals is even, the median is the arithmetical mean ofthe two middle values. Median survival time is computed on the basis ofthe entire population and there are no deletion of early deaths orsurvivors, with the following exception:

[1346] C. Computation of MedST from Survivors

[1347] If the total number of animals including survivors (N) is even,the MedST (days) (X+Y)/2, where X is the earlier day when the number ofsurvivors is N/2, and Y is the earliest day when the number of survivors(N/2)−1. If N is odd, the MedST (days) is X.

[1348] D. Computation of MedST from Mortality Distribution

[1349] If the total number of animals including survivors (N) is even,the MedST (days) (X+Y)/2, where X is the earliest day when thecumulative number of deaths is N/2, and Y is the earliest day when thecumulative number of deaths is (N/2)+1. If N is odd, the MedST (days) isX. “Cures” and “no-takes” in systems evaluated by MedST are based uponthe day of evaluation. On the day of evaluation any survivor notconsidered a “no-take” is recorded as a “cure.” Survivors on day ofevaluation are recorded as “cures” or “no-takes,” but not eliminatedfrom the calculation.

[1350] E. Calculation of Approximate Tumor Weight from Measurement ofTumor Diameters with Vernier Calipers

[1351] The use of diameter measurements (with Vernier calipers) forestimating treatment effectiveness on local tumor size permits retentionof the animals for lifespan observations. When the tumor is implantedsc, tumor weight is estimated from tumor diameter measurements asfollows. The resultant local tumor is considered a prolate ellipsoidwith one long axis and two short axes. The two short axes are assumed tobe equal. The longest diameter (length) and the shortest diameter(width) are measured with Vernier calipers. Assuming specific gravity isapproximately 1.0, and Pi is about 3, the mass (in mg) is calculated bymultiplying the length of the tumor by the width squared and dividingthe product by two. Thus,${{Tumor}\quad {weight}\quad ({mg})} = {\frac{{length}\quad ({mm}) \times \left( {{width}\quad\lbrack{mm}\rbrack} \right)^{2}}{2}\quad {or}\quad \frac{L \times (W)^{2}}{2}}$

[1352] The reporting of tumor weights calculated in this way isacceptable inasmuch as the assumptions result in as much accuracy as theexperimental method warrants.

[1353] F. Calculation of Tumor Diameters

[1354] The effects of a drug on the local tumor diameter may be reporteddirectly as tumor diameters without conversion to tumor weight. Toassess tumor inhibition by comparing the tumor diameters of treatedanimals with the tumor diameters of control animals, the three diametersof a tumor are averaged (the long axis and the two short axes). A tumordiameter T/C of 75% or less indicates activity and a T/C of 75% isapproximately equivalent to a tumor weight T/C of 42%.

[1355] G. Calculation of Mean Tumor Weight from Individual ExcisedTumors

[1356] The mean tumor weight is defined as the sum of the weights ofindividual excised tumors divided by the number of tumors. Thiscalculation is modified according to the rules listed below regarding“no-takes.” Small tumors weighing 39 mg or less in control mice or 99 mgor less in control rats, are regarded as “no-takes” and eliminated fromthe computations. In treated groups, such tumors are defined as“no-takes” or as true drug inhibitions according to the following rules:Percent of Percent of small tumors in “no-takes” in treated groupcontrol group Action ≦17 Any percent no-take; not used in calculations18-39 <10 drug inhibition; use in calculations ≧10 no-takes; not used incalculations ≧40 <15 drug inhibition; use in calculations ≧15 Code allnontoxic tests “33”

[1357] Positive control compounds are not considered to have “no-takes”regardless of the number of “no-takes” in the control group. Thus, thetumor weights of all surviving animals are used in the calculation ofT/C for the positive control (T/C defined above) SDs of the mean controltumor weight are computed the factors in a table designed to estimate SDusing the estimating factor for SD given the range (difference betweenhighest and lowest observation). Biometrik Tables for Statisticians(Pearson E S, and Hartley H G, eds.) Cambridge Press, vol. 1, table 22,p. 165.

[1358] II. Specific Tumor Models

[1359] A. Lymphoid Leukemia L1210

[1360] Summary: Ascitic fluid from donor mouse is transferred intorecipient BDF1 or CDF1 mice. Treatment begins 24 hours after implant.Results are expressed as a percentage of control survival time. Undernormal conditions, the inoculum site for primary screening is i.p., thecomposition being tested is administered i.p., and the parameter is meansurvival time. Origin of tumor line: induced in 1948 in spleen and lymphnodes of mice by painting skin with MCA. J Natl Cancer Inst. 13:1328,1953. Animals One sex used for all test and control animals in oneexperiment. Tumor Transfer Inject ip, 0.1 ml of diluted ascitic fluidcontaining 10⁵ cells Propagation DBA/2 mice (or BDF1 or CDF1 for onegeneration). Time of Transfer Day 6 or 7 Testing BDF₁ (C57BL/6 × DBA/2)or CDF₁ (BALB/c × DBA/2) Time of Transfer Day 6 or 7 Weight Within a 3-grange, minimum weight of 18 g for males and 17 g for females. Exp Size(n) 6/group; No. of control groups varies according to number of testgroups. Testing Schedule DAY PROCEDURE 0 Implant tumor. Preparematerials. Run positive control in every odd-numbered experiment. Recordsurvivors daily. 1 Weigh and randomize animals. Begin treatment withtherapeutic composition. Typically, mice receive 1 μg of the testcomposition in 0.5 ml saline. Controls receive saline alone. Treatmentis one dose/week. Any surviving mice are sacrificed after 4 wks oftherapy. 5 Weigh animals and record. 20 If there are no survivors exceptthose treated with positive control compound, evaluate 30 Kill allsurvivors and evaluate experiment.

[1361] Quality Control: Acceptable control survival time is 8-10 days.Positive control compound is 5-fluorouracil; single dose is 200mg/kg/injection, intermittent dose is 60 mg/kg/injection, and chronicdose is 20 mg/kg/injection. Ratio of tumor to control (T/C) lower limitfor positive control compound is 135%.

[1362] Evaluation: Compute mean animal weight on Days 1 and 5, and atthe completion of testing compute T/C for all test groups with >65%survivors on Day 5. A T/C value 85% indicates a toxic test. An initialT/C 125% is considered necessary to demonstrate activity. A reproducedT/C 125% is considered worthy of further study. For confirmed activity acomposition should have two multi-dose assays that produce a T/C 125%.

[1363] B. Lymphocytic Leukemia P388

[1364] Summary: Ascitic fluid from donor mouse is implanted in recipientBDF1 or CDF1 mice. Treatment begins 24 hours after implant. Results areexpressed as a percentage of control survival time. Under normalconditions, the inoculum site for primary screening is ip, thecomposition being tested is administered ip daily for 9 days, and theparameter is MedST. Origin of tumor line: induced in 1955 in a DBA/2mouse by painting with MCA. Scientific Proceedings, Pathologists andBacteriologists 33:603, 1957. Animals One sex used for all test andcontrol animals in one experiment. Tumor Transfer Inject ip, 0.1 ml ofdiluted ascitic fluid containing 10⁶ cells Propagation DBA/2 mice (orBDF1 or CDF1 for one generation). Time of Transfer Day 7 Testing BDF₁(C57BL/6 × DBA/2) or CDF₁ (BALB/c × DBA/2) Time of Transfer Day 6 or 7Weight Within a 3-g range, minimum weight of 18 g for males and 17 g forfemales. Exp Size (n) 6/group; No. of control groups varies according tonumber of test groups. Testing Schedule DAY PROCEDURE 0 Implant tumor.Prepare materials. Run positive control in every odd-numberedexperiment. Record survivors daily. 1 Weigh and randomize animals. Begintreatment with therapeutic composition. Typically, mice receive 1 ng ofthe test composition in 0.5 ml saline. Controls receive saline alone.Treatment is one dose/week. Any surviving mice are sacrificed after 4wks of therapy. 5 Weigh animals and record. 20 If there are no survivorsexcept those treated with positive control compound, evaluate 30 Killall survivors and evaluate experiment.

[1365] Acceptable MedST is 9-14 days. Positive control compound is5-fluorouracil: single dose is 200 mg/kg/injection, intermittent dose is60 mg/kg/injection, and chronic dose is 20.mg/kg/injection. T/C lowerlimit for positive control compound is 135% Check control deaths, notakes, etc.

[1366] Quality Control: Acceptable MedST is 9-14 days. Positive controlcompound is 5-fluorouracil: single dose is 200 mg/kg/injection,intermittent dose is 60 mg/kg/injection, and chronic dose is 20mg/kg/injection. T/C lower limit for positive control compound is 135%.Check control deaths, no takes, etc.

[1367] Evaluation: Compute mean animal weight on Days 1 and 5, and atthe completion of testing compute T/C for all test groups with >65%survivors on Day 5. A T/C value of 85% indicates a toxic test. Aninitial T/C of 125% is considered necessary to demonstrate activity. Areproduced T/C 125% is considered worthy of further study. For confirmedactivity a composition should have two multi-dose assays that produce aT/C 125%.

[1368] C. Melanotic Melanoma B16

[1369] Summary: Tumor homogenate is implanted ip or sc in BDF1 mice.Treatment begins 24 hours after either ip or sc implant or is delayeduntil an sc tumor of specified size (usually approximately 400 mg) canbe palpated. Results expressed as a percentage of control survival time.The composition being tested is administered ip, and the parameter ismean survival time. Origin of tumor line: arose spontaneously in 1954 onthe skin at the base of the ear in a C57BL/6 mouse. Handbook onGenetically Standardized Jax Mice. Jackson Memorial Laboratory, BarHarbor, Me., 1962. See also Ann NY Acad Sci 100, Parts 1 and 2, 1963.Animals One sex used for all test and control animals in one experiment.Propagation Strain C57BL/6 mice Tumor Transfer Implant fragment sc bytrochar or 12-g needle or tumor homogenate* every 10-14 days intoaxillary region with puncture in inguinal region. Testing Strain BDF₁(C57BL/6 × DBA/2) Time of Transfer Excise sc tumor on Day 10-14 fromdonor mice and implant as above Weight Within a 3-g range, minimumweight of 18 g for males and 17 g for females. Exp Size (n) 10/group;No. of control groups varies according to number of test groups. *Tumorhomogenate: Mix 1 g or tumor with 10 ml of cold balanced salt solution,homogenize, and implant 0.5 ml of tumor homogenate ip or sc. Fragment: A25-mg fragment may be implanted sc. Testing Schedule DAY PROCEDURE 0Implant tumor. Prepare materials. Run positive control in everyodd-numbered experiment. Record survivors daily. 1 Weigh and randomizeanimals. Begin treatment with therapeutic composition. Typically, micereceive 1 μg of the test composition in 0.5 ml saline. Controls receivesaline alone. Treatment is one dose/week. Any surviving mice aresacrificed after 8 wks of therapy. 5 Weigh animals and record. 60 Killall survivors and evaluate experiment.

[1370] Quality Control: Acceptable control survival time is 14-22 days.Positive control compound is 5-fluorouracil: single dose is 200mg/kg/injection, intermittent dose is 60 mg/kg/injection, and chronicdose is 20 mg/kg/injection. T/C lower limit for positive controlcompound is 135% Check control deaths, no takes, etc.

[1371] Evaluation: Compute mean animal weight on Days 1 and 5, and atthe completion of testing compute T/C for all test groups with >65%survivors on Day 5. A T/C value of 85% indicates a toxic test. Aninitial T/C of 125% is considered necessary to demonstrate activity. Areproduced T/C 125% is considered worthy of further study. For confirmedactivity a composition should have two multi-dose assays that produce aT/C 125%.

[1372] Metastasis after IV Injection of Tumor Cells

[1373] 10⁵ B16 melanoma cells in 0.3 ml saline are injectedintravenously in C57BL/6 mice. The mice are treated intravenously withIg of the composition being tested in 0.5 ml saline. Controls receivesaline alone. The treatment is given as one dose per week. Micesacrificed after 4 weeks of therapy, the lungs are removed andmetastases are enumerated.

[1374] C. 3LL Lewis Lung Carcinoma

[1375] Summary: Tumor may be implanted sc as a 2-4 mm fragment, or im asa 2×10⁶-cell inoculum. Treatment begins 24 hours after implant or isdelayed until a tumor of specified size (usually approximately 400 mg)can be palpated. The composition being tested is administered ip dailyfor 11 days and the results are expressed as a percentage of thecontrol. Origin of tumor line: arose spontaneously in 1951 as carcinomaof the lung in a C57BL/6 mouse. Cancer Res 15:39, 1955. See, alsoMalave, I. et al., J. Nat'l. Canc. Inst. 62:83-88 (1979). Animals Onesex used for all test and control animals in one experiment. PropagationStrain C57BL/6 mice Tumor Transfer Inject cells im in hind leg orimplant fragment sc in axillary region with puncture in inguinal region.Transfer on day 12-14 Testing Strain BDF₁ (C57BL/6 × DBA/2) or C3H miceTime of Transfer Same as above Weight Within a 3-g range, minimum weightof 18 g for males and 17 g for females. Exp Size (n) 6/group for scimplant, or 10/group for im implant.; No. of control groups variesaccording to number of test groups. Testing Schedule DAY PROCEDURE 0Implant tumor. Prepare materials. Run positive control in everyodd-numbered experiment. Record survivors daily. 1 Weigh and randomizeanimals. Begin treatment with therapeutic composition. Typically, micereceive 1 μg of the test composition in 0.5 ml saline/Controls receivesaline alone. Treatment is one dose/week. Any surviving mice aresacrificed after 4 wks of therapy. 5 Weigh animals and record. Final dayKill all survivors and evaluate experiment.

[1376] Quality Control: Acceptable im tumor weight on Day 12 is 500-2500mg. Acceptable im tumor MedST is 18-28 days. Positive control compoundis cyclophosphamide: 20 mg/kg/injection, qd, Days 1-11. Check controldeaths, no takes, etc.

[1377] Evaluation: Compute mean animal weight when appropriate, and atthe completion of testing compute T/C for all test groups. When theparameter is tumor weight, a reproducible T/C of 42% is considerednecessary to demonstrate activity. When the parameter is survival time,a reproducible T/C of 125% is considered necessary to demonstrateactivity. For confirmed activity a composition must have two multi-doseassays

[1378] D. 3LL Lewis Lung Carcinoma Metastasis Model

[1379] This model has been utilized by a number of investigators. See,for example, Gorelik, E. et al., J. Nat'l. Canc. Inst. 65:1257-1264(1980); Gorelik, E. et al., Rec. Results Canc. Res. 75:20-28 (1980);Isakov, N. et al., Invasion Metas. 2:12-32 (1982) Talmadge J. E. et al.,J. Nat'l. Canc. Inst. 69:975-980 (1982); Hilgard, P. et al., Br. J.Cancer 35:78-86(1977)).

[1380] Mice: male C57BL/6 mice, 2-3 months old. Tumor: The 3LL LewisLung Carcinoma was maintained by sc transfers in C57BL/6 mice. Followingsc, im or intra-footpad transplantation, this tumor produces metastases,preferentially in the lungs. Single-cell suspensions are prepared fromsolid tumors by treating minced tumor tissue with a solution of 0.3%trypsin. Cells are washed 3 times with PBS (pH 7.4) and suspended inPBS. Viability of the 3LL cells prepared in this way is generally about95-99% (by trypan blue dye exclusion). Viable tumor cells (3×10⁴-5×10⁶)suspended in 0.05 ml PBS are injected into the right hind foot pads ofC57BL/6 mice. The day of tumor appearance and the diameters ofestablished tumors are measured by caliper every two days. Typically,mice receive 1 μg of the composition being tested in 0.5 ml saline.Controls receive saline alone. The treatment is given as one or twodoses per week.

[1381] In experiments involving tumor excision, mice with tumors 8-10 mmin diameter are divided into two groups. In one group, legs with tumorsare amputated after ligation above the knee joints. Mice in the secondgroup are left intact as nonamputated tumor-bearing controls. Amputationof a tumor-free leg in a tumor-bearing mouse has no known effect onsubsequent metastasis, ruling out possible effects of anesthesia, stressor surgery. Surgery is performed under Nembutal anesthesia (60 mgveterinary Nembutal per kg body weight).

[1382] Determination of Metastasis Spread and Growth

[1383] Mice are killed 10-14 days after amputation. Lungs are removedand weighed. Lungs are fixed in Bouin's solution and the number ofvisible metastases is recorded. The diameters of the metastases are alsomeasured using a binocular stereoscope equipped with amicrometer-containing ocular under 8× magnification. On the basis of therecorded diameters, it is possible to calculate the volume of eachmetastasis. To determine the total volume of metastases per lung, themean number of visible metastases is multiplied by the mean volume ofmetastases. To further determine metastatic growth, it is possible tomeasure incorporation of ¹²⁵IdUrd into lung cells (Thakur, M. L. et al.,J. Lab. Clin. Med. 89:217-228 (1977). Ten days following tumoramputation, 25 mg of FdUrd is inoculated into the peritoneums oftumor-bearing (and, if used, tumor-resected mice. After 30 min, mice aregiven 1 mCi of ¹²⁵IdUrd. One day later, lungs and spleens are removedand weighed, and a degree of ¹²⁵IdUrd incorporation is measured using agamma counter.

[1384] Statistics: Values representing the incidence of metastases andtheir growth in the lungs of tumor-bearing mice are not normallydistributed. Therefore, non-parametric statistics such as theMann-Whitney U-Test may be used for analysis.

[1385] Study of this model by Gorelik et al. (1980, supra) showed thatthe size of the tumor cell inoculum determined the extent of metastaticgrowth. The rate of metastasis in the lungs of operated mice wasdifferent from primary tumor-bearing mice. Thus in the lungs of mice inwhich the primary tumor had been induced by inoculation of large dosesof 3LL cells (1-5×10⁶) followed by surgical removal, the number ofmetastases was lower than that in nonoperated tumor-bearing mice, thoughthe volume of metastases was higher than in the nonoperated controls.Using ¹²⁵IdUrd incorporation as a measure of lung metastasis, nosignificant differences were found between the lungs of tumor-excisedmice and tumor-bearing mice originally inoculated with 10⁶ ³LL cells.Amputation of tumors produced following inoculation of 10⁵ tumor cellsdramatically accelerated metastatic growth. These results were in accordwith the survival of mice after excision of local tumors. The phenomenonof acceleration of metastatic growth following excision of local tumorshad been observed by other investigators. The growth rate and incidenceof pulmonary metastasis were highest in mice inoculated with the lowestdoses (3×10⁴-10⁵ of tumor cells) and characterized also by the longestlatency periods before local tumor appearance. Immunosuppressionaccelerated metastatic growth, though nonimmunologic mechanismsparticipate in the control exerted by the local tumor on lung metastasisdevelopment. These observations have implications for the prognosis ofpatients who undergo cancer surgery.

[1386] E. Walker Carcinosarcoma 256

[1387] Summary: Tumor may be implanted sc in the axillary region as a2-6 mm fragment, im in the thigh as a 0.2-ml inoculum of tumorhomogenate containing 10⁶ viable cells, or ip as a 0.1-ml suspensioncontaining 10⁶ viable cells. Treatment of the composition being testedis usually ip. Origin of tumor line: arose spontaneously in 1928 in theregion of the mammary gland of a pregnant albino rat. J Natl Cancer Inst13:1356, 1953. Animals One sex used for all test and control animals inone experiment. Propagation Strain Random-bred albino Sprague-Dawleyrats Tumor Transfer S.C. fragment implant is by trochar or 12-g needleinto axillary region with puncture in inguinal area. I.m. implant iswith 0.2 ml of tumor homogenate (containing 10⁶ viable cells) into thethigh. I.p. implant is with 0.1 ml suspension (containing 10⁶ viablecells) Day 7 for im or ip implant; Days 11-13 for sc implant TestingStrain Fischer 344 rats or random-bred albino rats Time of Transfer Sameas above Weight 50-70 g (maximum of 10-g weight range within eachexperiment) Exp Size (n) 6/group; No. of control groups varies accordingto number of test groups. Prepare drug Administer Weigh animals Evaluateon Test system on day: drug on days: on days days 5WA16 2 3-6 3 and 7 75WA12 0 1-5 1 and 5 10-14 5WA31 0 1-9 1 and 5 30

[1388] In addition the following general schedule is followed DAYPROCEDURE 0 Implant tumor. Prepare materials. Run positive control inevery odd-numbered experiment. Record survivors daily. 1 Weigh andrandomize animals. Begin treatment with therapeutic composition.Typically, mice receive 1 μg of the test composition in 0.5 ml saline.Controls receive saline alone. Treatment is one dose/week. Any survivingmice are sacrificed after 4 wks of therapy. Final day Kill all survivorsand evaluate experiment.

[1389] Quality Control: Acceptable i.m. tumor weight or survival timefor the above three test systems are: 5WA16: 3-12 g.; 5WA12: 3-12 g.;5WA31 or 5WA21: 5-9 days.

[1390] Evaluation: Compute mean animal weight when appropriate, and atthe completion of testing compute T/C for all test groups. When theparameter is tumor weight, a reproducible. T/C 42% is considerednecessary to demonstrate activity. When the parameter is survival time,a reproducible T/C 125% is considered necessary to demonstrate activity.For confirmed activity.

[1391] F. A20 lymphoma

[1392] 10⁶ murine A20 lymphoma cells in 0.3 ml saline are injectedsubcutaneously in Balb/c mice. The mice are treated intravenously withIg of the composition being tested in 0.5 ml saline. Controls receivesaline alone. The treatment is given as one dose per week. Tumor growthis monitored daily by physical measurement of tumor size and calculationof total tumor volume. After 4 weeks of therapy the mice are sacrificed.

[1393] Treatment Regimens and Results (Constructs)

[1394] For determining efficacy in the tumor models described above thegeneral categories of therapeutic constructs used are given below. Forall of the classes of conjugates listed below, the SAg component can beprepared as either a DNA encoding SAg or as the SAg polypeptide itselfIn either form the SAg DNA or protein may be conjugated to additionalmolecules, either nucleic acid or polypeptides. Operationally, fortherapeutic use in vivo or ex vivo, these conjugates may be prepared bychemical coupling or by recombinant means (whichever is appropriate) andconjugated to a tumor-targeting structure or incorporated into a vehicle(e.g., liposomes) that themselves comprise a tumor targetingstructure(s). Again, examples of such targeting structures include, butare not limited to, an antibody, antigen, receptor or receptor ligand.Methods are disclosed in Examples 1, 3, 4, 5, 6, 7, 14, 17, 18, 30-32.

[1395] 1. SAg Nucleic Acid Constructs including Phage Displays and SAgTransfected Bacterial Cells

[1396] 2. Glycosylated SAgs

[1397] 3. Chimeric SAgs

[1398] Conjugates having a Superantigen component (polypeptide ornucleic acid) and a partner that is either a single component or aconjugate of 2 or more components (protein, carbohydrate, lipid or DNA)as indicated below: Superantigen (Protein or DNA) Partner (SingleComponent or Coniuqate)  4. DNA coding sequence  5. Polypeptide  6.Nucleic acid  7. Tumor associated Peptide  8. Tumor Antigen-MHC protein 9. LPS 10. Lipoarabinomannan 11. Ganglioside 12. Glycosphingolipid 13.Ganglioside-CD1 receptor 14. Glycosphingolipid-CD1 receptor 15.Glycosylceramide (e.g., Gal-Cer) 16. GalCer-CD1 receptor 17. Gal 18.Arg-Gly-Asp or Asn-Gly-Arg 19 iNOS 20. Gb2 or Gb3 or Gb4 21. (Gb2 or Gb3or Gb4)-CD1 receptor 22. -GPI-(Gb2 or Gb3 or Gb4) 23. -GPI-(Gb2 or Gb3or Gb4)-CD1 receptor 24. Verotoxin 25. Verotoxin A or B Subunit 26. IFN□receptor peptide homologous to VT 27. CD19 peptide homologous to VT 28.LDL, VLDL, HDL, IDL 29. Apolipoproteins (e.g., Lp(a), apoB-100, apoB-48,apoE) 30. OxyLDL, oxyLDL mimics, (e.g., 7β-hydroperoxycholesterol,7□-hydroxycholesterol, 7- ketocholesterol, 5□-6□-epoxycholesterol,7□-hydroperoxy-choles-5-en-3β-ol, 4- hydroxynonenal (4-HNE), 9-HODE,13-HODE and cholesterol-9-HODE) 31. OxyLDL by products (e.g.lysolecithin, lysophosphatidylcholine, malondialdehyde, 4-hydroxynonenal) 32. LDL & oxyLDL receptors (e.g., LDL oxyLDL,acetyl-LDL, VLDL, LRP, CD36, SREC, LOX-1, macrophage scavengerreceptors) 33. phytosphingosine, -GPI-phytosphingosine 34. tumorassociated lipid antigens 35. glycolipid, proteolipid,glycosphingolipid, sphingolipid with inositolphosphate -containing headgroups, phytoglycolipids, mycoglycolipids, -GPI-sphingosines,-GPI-lipids 36. sphingolipids with inositolphosphate-containing headgroups having the general structure: ceramide-P-myoinositol-X with Xreferring to polar substituents comprising ceramide-p-inositol-mannose,inositol-1-P-(6)mannose(a1,2 inositol-1P-(1)ceramide,(inositol-P)2-ceramide, inositol-P-inositol-P-ceramide,inositol-P-inositol-P-ceramide. 37. tumor associated glycan antigensconsisting of peptidoglycans or glycan phosphotidyinositol (GPI)structures

[1399] Vaccine Use

[1400] For use as a vaccine, the constructs are administeredsubcutaneously, intramuscularly intradermally or intraperitoneally indoses ranging from 50 to 500 ng in various vehicles such as Freund'sadjuvant, aluminum hydroxide, pluruonic acid triblock and liposomes asdescribed in the art. Doses may be repeated every 10 days. Tumors areimplanted after the last dose. A control group does not receive thevaccine.

[1401] Use in Established Tumors

[1402] For proteins or nucleic acid constructs, treatment consists ofinjecting animals iv or ip with 50, 500 1000 or 5,000 ng of in 0.1-0.5ml of normal saline. Unless indicated otherwise above, treatments aregiven one to three times per week for two to five weeks. Phage displays,yeast displays and vesilcle, SAg-bacterial or viral constructs or SAgvesicles are administered as 10⁹ transducing units (TU) and irradiatedbacterial cells, yeast cells as 10⁵-10⁶ cells iv into the tail vein oneto three times per week for two to five weeks or directly into tumor in30-75% or the iv doses on the same schedule. Exosomes or vesicles,harvested from transfected, transformed or fusion tumor cells or sickledcells or mutant yeast are given i.v. into the tail vein in a dose of0.25-1 g per animal one to three times per week for two to five weeks.The results shown in Table VI are for each composition and dose tested.The results are statistically significant by the Wilcoxon rank sum test.

[1403] Treatment regimens for SAg activated effector T or NKT cells arein Example 16, 18, 19. The preferred animal model for evaluation of theadoptively transferred T or NKT effector cells is the MCA 205/207fibrosarcoma with pulmonary metastases (Shu S. et al., J. Immunol. 152:1277-1288 (1994)). The other models given in Example 20 are alsosuitable for evaluation of the therapeutic effectiveness of the effectorT cells. TABLE VI Tumor Model Parameter % of Control Response L1210MST >130%  P388 MST >130%  B16 MST >130%  B16 metastasis Median numberof metastases <70% 3LL MST >130%  Mean tumor weight <40% 3LL metastasisMST >130%  Mean lung weight <60 Median number of metastases <60% Medianvolume of metastases <60% Medial volume of metastases <60% Median uptakeof IdUrd <60% Walker carcinoma MedST >130%  Mean tumor weight <40% A20MST >130%  Mean tumor volume <40%

EXAMPLE 22 Antitumor Effects of Therapeutic Constructs and Effector T,NKT Cells or Sickled Erythrocytes in Human Patients

[1404] All patients treated have histologically confirmed malignantdisease including carcinomas, sarcomas, melanomas, lymphomas andleukemia and have failed conventional therapy. Patients may be diagnosedas having any stage of metastatic disease involving any organ system.Staging describes both tumor and host, including organ of origin of thetumor, histologic type and histologic grade, extent of tumor size, siteof metastases and functional status of the patient. A generalclassification includes the known ranges of Stage I (localized disease)to Stage 4(widespread metastases). Patient history is obtained andphysical examination performed along with conventional tests ofcardiovascular and pulmonary function and appropriate radiologicprocedures. Histopathology is obtained to verify malignant disease.

EXAMPLE 23 Treatment Procedures

[1405] Constructs (or Preparations)

[1406] Doses of the constructs are determined as described above using,inter alia, appropriate animal models of tumors. Two classes oftherapeutic compositions are administered namely SAg proteins or SAgconjugates (nucleic acids or peptides-polypeptides), SAg phage displays,SAg yeast displays, SAg bacterial cell displays, as described above foranimal models.

[1407] A treatment consists of injecting the patient with 0.5-500 mg ofConstruct intravenously in 200 ml of normal saline over a one hourperiod. Treatments are given 3×/week for a total of 12 treatments.Patients with stable or regressing disease are treated beyond the12^(th) treatment. Treatment is given on either an outpatient orinpatient basis as needed.

[1408] Effector T or NKT Cells

[1409] Eligible patients are treated with tumor antigens such asirradiated tumor cells or GM-CSF transduced tumor cells injectedapproximately 10 centimeters from a draining lymph node site. Ten dayspost injection, draining lymph nodes are obtained in a limited surgicalprocedure at the site draining the injection. The lymph nodes areconverted to a single cell suspension of lymphocytes and these areincubated with various SAg preparations for two days followed by Il-2for an additional 72 hours. These lymphocytes now called effector Tcells or NKT cell are used for adoptive immunotherapy.

[1410] Effector T or NKT cells harvested by centrifugation at 500×g for15 min and the cell pellets are pooled. After washing the cells in HBSS,the cell are resuspended in 200 ml of normal saline containing 5% humanserum albumin and 450,000 IU of IL-2 for transfer. Each recipient willreceive four escalating doses or 33 million, 100 million, 330 millionand 1 billion cells per square meter of body surface area each given oneweek apart. Cells are infused through a subclavian central venouscatheter over a 30- minute interval. IL-2 administration i.v. iscommenced immediately after completion of cell infusion at a dose andschedule of 180,000 IU/ml every 8 h. for 5 days. All patients receiveindomethacin (50 mg P.O.) every 8 h, acetaminophen (650 mg P.O.) every 6h. and ranitidine (150 mg P.O.) every 12 h while receiving IL-2 in orderto reduce febrile and gastric side effects. As controls, a cohort ofpatients is treated with the in vivo tumor vaccination step and IL-2without the tumor effector cells. Patients will be followed for clinicalresponse every 4 weeks for 2 months with repeat radiologicalexaminations.

[1411] Abbreviated Exemplary Human Protocol: Sequential Administrationof GM-CSF Transduced Tumor Cells In Vivo and SAg Activated NKT and TCells Ex Vivo in Patients with Metastatic Renal Cell Carcinoma andMelanoma

[1412] In vivo Phase: Immunization with GM-CSF Transduced Tumor Cells

[1413] Day 1: GM CSF transfected tumor cells (renal carcinoma/melanoma)are injected as given in Phase I GM-CSF Gene Transduction Protocol[Human Gene Therapy 6: 347-368, (1995)]

[1414] Day 7-10: Lymph Nodes draining the GM-CSF transfected tumor cellsites are removed and placed in tissue culture OR patients are pheresedand their peripheral blood T cells and NKT cells collected for furthertreatment in tissue culture as described below.

[1415] Ex Vivo Phase: Immunization with SAg

[1416] 1. The T cells are obtained from either lymph nodes drainingGM-CSF transduced tumor cell immunization or peripheral blood andsubdivided into CD4+CD8+ (T cell) and CD4-CD8− (NKT cell) populations.

[1417] 2. SAg enterotoxin B is added to cultures of the NKT and T cellpopulations for 48 hours.

[1418] 3. The NKT cells and T cells are further expanded for anadditional 72 hours (optional).

[1419] SAg Activated NKT and/or T Cell Administration

[1420] 1. The CD4+CD8+ (T cell) and CD4-CD8− (NKT) populations areharvested for injection into patients.

[1421] 2. T cells or NKT cells are administered with a mean 1011 cellsper patient.

[1422] Assessment:

[1423] 1. T cells phenotypes for NKT cell markers, V expression, CD44,CD62 are carried out on lymph node and peripheral blood T cells or NKTcells immediately after their removal and at various intervals after exvivo SAg stimulation and expansion.

[1424] 2. Tumor and DTH assessment are as described in the Phase IProtocol on GM-CSF Transduction [Human Gene Therapy 6: 347-368 (1995)].

[1425] Patient Evaluation

[1426] Assessment of response of the tumor to the therapy is made onceper week during therapy and 30 days thereafter. Depending on theresponse to treatment, side effects, and the health status of thepatient, treatment is terminated or prolonged from the standard protocolgiven above. Tumor response criteria are those established by theInternational Union Against Cancer and are listed in Table VII. TABLEVII RESPONSE DEFINITION Complete remission (CR) Disappearance of allevidence of disease Partial remission (PR) >50% decrease in the productof the two greatest perpendicular tumor diameters; no new lesions Lessthan partial remission 25-50% decrease in tumor size, (<PR) stable forat least 1 month Stable disease <25% reduction in tumor size; noprogression or new lesions Progression >25% increase in size of any onemeasured lesion or appearance of new lesions despite stabilization orremission of disease in other measured sites

[1427] The efficacy of the therapy in a population is evaluated usingconventional statistical methods including, for example, the Chi Squaretest or Fisher's exact test. Long-term changes in and short term changesin measurements can be evaluated separately.

[1428] Results: One hundred and fifty patients are treated. The resultsare summarized in Table VIII. Positive tumor responses are observed in80% of the patients as follows: TABLE VIII All Patients Response No. %PR 20 66% <PR 10 33% Tumor Types Response % of Patients BreastAdenocarcinoma PR +< PR 80% Gastrointestinal Carcinoma PR +< PR 75% LungCarcinoma PR +< PR 75% Prostate Carcinoma PR +< PR 75% Lymphoma/LeukemiaPR +< PR 75% Head and Neck Cancer PR +< PR 75% Renal and Bladder CancerPR +< PR 75% Melanoma PR +< PR 75%

EXAMPLE 24 Preparation of DCs

[1429] Splenocytes obtained from naive C57BL/6 females are treated withammonium chloride Tris buffer for 3 min at 37° C. to deplete red bloodcells. Splenocytes (3 ml) at 2×10⁷ cells/ml are layered over 2 mlmetrizamide gradient column (Nycomed Pharma AS, Oslo, Norway; analyticalgrade, 14.5 g added to 100 ml PBS, pH 7.0) and centrifuged at 600 g for10 mm. The DC-enriched fraction from the interface is further enrichedby adherence for 90 mm. Adherent cells (mostly DC and a fewcontaminating macrophages) are retrieved by gentle scraping andsubjected to a second round of adherence at 37° C. for 90 min to depletethe contaminating macrophages. Non-adherent cells are pooled as splenicDC, and by FACS® analysis are ˜80-85% DC (stainwith mAb 33D1), 1-2%macrophages (stain with mAb F4/80), 10% T cells, and <5% B cells. Thepellet is resuspended and enriched for macrophages by two rounds ofadherence at 37° C. for 90 mm each. More than 80% of the adherentpopulation is identified as macrophages by FACS® analysis with 5%lymphocytes and <5% DC. B cells are separated from the non-adherentpopulation (B and T cells) by panning on anti-Ig-coated plates. Theseparated cell population which is comprised of >80% T lymphocytes byFACS analysis is used as responder T cells.

[1430] Generation of Bone Marrow-Derived DCs

[1431] Erythrocyte depleted mouse bone marrow cells from flushed marrowcavities are cultured in CM with 10 ng/ml GM-CSF and 10 ng/ml IL-4 at106 cells/ml. On day 7, DCs are harvested by gentle pipetting and areenriched by 14.5% (by weight) metrizamide (Sigma) CM gradients. The lowdensity interface containing the DC is collected by gentle pipetteaspiration. The floating DCs express CD11b, CD11c, CD86, DEC205, MHCclass I and II and CD40. They are negative or low for CD3 and B220expression.

[1432] DC Cultures

[1433] Mouse BM-DCs are prepared in CM with IL-4 and GM-CSF (1000 IU/mleach). The DC are washed twice with CM, enumerated purity >90% bypositive coexpression of MHC class II, CD40, CD80, CD86, and CD11c byfluorescence-activated cell sorter (ACS)], and cultured in CM with addedcytokines for further studies. Human-monocyte-derived DCs are obtainedfrom the adherent fraction of mononuclear cells of healthy volunteersand are incubated 7-8 days in AIMV containing L-Glu, antibiotics andrhIL-4 and rhGM-CSF (1000 IU/ml each, Schering Plough, Kenilworth, N.J.,USA). After 8 days in culture, the loosely adherent or floating cellsshow typical dendritic morphology, express high levels of MHC class Iand II molecules, CD40 and CD86; most are positive for CD1a and CD11cbut low or negative for CD2, CD3, CD14, CD19 and CD83.

EXAMPLE 25 Preparation of DC/Tumor Cells Hybrids (DC/tc)

[1434] DCs derived from BM culture are fused with tumor cells at a 3:1(DC:tumor cell) ratio using polyethylene glycol (PEG; MW=1450)/DMSOsolution (Sigma). In brief, tumor cells are cultured in CM supplementedwith 20% FCS and 1×OPI solution (oxaloacetate, pyruvate, and insulin;Sigma) for 4-6 h before fusion. Tumor cells and DCs are then mixed andwashed with serum-free medium. After removing the medium, 1 ml of PEG isadded to the cell pellet while resuspending the cells by stirring for 2min. An additional 10 ml of serum-free medium is added to the cellsuspension over the next 3 min. with continued stirring. The cells arecentrifuged at 400×g for 5 mm. The cells are resuspended with 20% FCS CMand cultured for 24 h before staining or being used as targets orvaccines. Fusion preparations of DCs with B16 or RMA-S are termed B16/DCand RMA-S/DC, respectively.

[1435] Phenotype Staining of Fused Hybrid Cells

[1436] B16, RMA-S, DCs, and their fused hybrids are analyzed by stainingwith FITC- or PE-conjugated mAbs (PharMingen) against MHC antigens(D^(b), K^(b), IA^(b)) adhesion and costimulatory molecules (B7.1,ICAM-1) and lymphocyte antigens (Thy-1.2, SmIg) at 4° C. for 45 min. DCswere identified by labeling with mAb against CD11c (N418). B16, B16/DCor B16/B16 fused cells are stained with mAb against AKV Env gp85 protein(M562, provided by Dr. Masaru Taniguchi, Chiba University, Tokyo, Japan)as a B16 tumor-specific marker. RMA-S and RMA-S/DC fused cells arestained with Thy-1.2 or mAb against the R-MuLV-encoded Gag p12 protein(584, provided by Dr. Bruce Chesebro, National Institute of Allergy andInfectious Diseases, Hamilton, Mo.) as RMA-S tumor-derived markers. Themethod for labeling cells with TRITC (rhodamine) is described. Briefly,cells are resuspended in RPMI 1640 at 106 cells/ml and incubated withTR1TC (0.5g/ml) in 37° C. for 45 mm. The labeled cells are washed threetimes and used for fusion studies. The phenotypes of fresh and culturedLN T cells is determined by FACS analysis following staining with FITC-or PE-conjugated mAbs against Thy-1.2, Lyt-2, and L3T4 (PharMingen). Allcells are washed twice with HBSS and fixed with 0.2% paraformaldehyde.Fluorescence intensity and positive cell percentage were measured on aFACScan flow microfluorometer (Becton Dickinson, Sunnyvale, Calif.).

[1437] Additional Fusion Methods

[1438] Murine (CS 7BL16) MC38 adenocarcinoma cells are stablytransfected with the DF3/MUCI cDNA (MC38/MUC1). MC38, MC38/MUC1 and thesyngeneic MB49 bladder cancer cells are maintained in DMEM supplementedwith 10% heat-inactivated fetal calf serum (FCS), 2 mM glutamine, 100U/ml penicillin and 100 mg/ml streptomycin. DCs are obtained asdescribed from bone marrow culture with certain modifications. Briefly,bone marrow is flushed from long bones, and red cells are lysed withammonium chloride. Lymphocytes, granulocytes and Ia⁺ cells are depletedfrom the bone marrow cells by incubation with the following mAbs: (1)2.43, anti-CD8 (TIB 210; American Type Culture Collection, Rockville,Md.); (2) GK1.5, anti-CD4 (TIB 207); (3) RA3-3A1/6.1, anti B220/CD45R(TIB 146); (4) B21-2, anti-Ia (TIB 229); and (5) RB6-8C5, anti-Gr-1(PharMingen, San Diego, Calif.) and then rabbit complement. The cellsare plated in six-well culture plates in RPMI 1640 medium supplementedwith 5% heat-inactivated FCS, 50 M 2-mercaptoethanol, 1 mM HEPES (pH7.4), 2 mM glutamine, 10 U/ml penicillin, 100 mg/ml streptomycin and 500U/ml recombinant murine GM-CSF (Boehringer Mannheim, Indianapolis,Ind.). At day 7 of culture, nonadherent and loosely adherent cells arecollected and replated in 100-mm petri dishes (10⁶ cells/ml; 8 ml/dish).The nonadherent cells are washed away after 30 mm of incubation, andGM-CSF in RPMI medium is added to the adherent cells. After 18 h, thenonadherent cell population is removed for fusion with MC38/MUC1 orMC38. Fusion is carried out with 50% PEG in Dulbecco's PBS without Ca²⁺or Mg²⁺ at pH 7.4. The fused cells are plated in 24-well culture platesin the presence of HAT medium (Sigma) for 10-14 days. HAT slowsproliferation of MC38/MUC1 and MC38, but not the fused cells. MC38/MUC1and MC38 cells grow firmly attached to the tissue culture flask, whilethe fused cells are dislodged by gentle pipetting.

[1439] Flow Cytometry

[1440] Cells are washed with PBS and incubated with mAb DF3 (anti-MUCI),mAb M1/42/3.9.8 (anti-MHC class I), mAb M5/114 (anti-MHC class II), mAb16-IOA1 (anti-B7-1), mAb GL1 (anti-B7-2) or mAb 3E, (anti-ICAM-1) for 30mm on ice. After washing with PBS, the appropriate fluoresceinisothiocyanate (FITC)-conjugated anti-hamster, -rat and -mouse IgG isadded for another 30 mm on ice. Samples are then washed, fixed andanalyzed in a FACScan (Becton Dickinson, Mountain View, Calif.).

[1441] Fusion of SAg Transfected Tumor Cells with Dendritic CellsPreparation of Dendritic Cells

[1442] Dendritic cells were generated from mouse bone marrow cultures ofB6 mouse origin, following the protocol provided by W. Storkus, et al.Bone marrow cells were prepared from the femurs of four 8 week oldnormal C576L/6 (denoted B6, H 2^(b)) mice.

[1443] 1. For all steps, a complete medium consisting of RPMI 1640 mediasupplemented with 10% heat-inactivated FCS 0.1 mM NEAA 1 mM sodiumpyruvate, 50 mM 2-mercaptoethanol, 50 mM HEPES, 2 mM glutamine, 100 U/mlpenicillin, and 100 U/ml streptomycin was used.

[1444] 2. Following removal of both femurs from each of four mice, cellswere extruded by use of a 3 cc syringe filled with complete medium and25 G needle. Clumps were removed by passing the cell suspension througha Cell Strainer (Falcon 2350). The cell suspension was then centrifugedat 600×g for 5 minutes at room temperature (all further centrifugationswere performed in this manner unless otherwise indicated).

[1445] 3. Red blood cells were lysed by resuspending the cells in 4 mlof Red Blood Cell Lysing Solution (Sigma R7757) and incubating the tubeon ice for 2 minutes. After neutralizing the ammonium chloride actionwith complete medium, the cells were centrifuged to pellet them.

[1446] 4. Granulocytes and leukocytes were depleted from the bone marrowcells by incubation in 2 ml of a cocktail of monoclonal antibodies fromPharmingen, formulated to contain no azide and have low endotoxinlevels. The cocktail consisted of 5 mg/ml of the following monoclonalantibodies (1) 2.43, anti-CDS, Pharmingen 01050D (2) GK 15, anti-CD4,Pharmingen 094200 (3) RA3-3A1/6. 1, anti-B220/CD45R, Pharmingen 01120D(4) anti GRI, Pharmingen 01210D. Antibodies were diluted in completemedium, cells were then resuspended in the cocktail and incubated forone hour on ice. After diluting the cell suspension to 30 ml withcomplete medium, the cells were centrifuged to pellet them.

[1447] 5. Cells were then resuspended in 12 ml of a 1:8 dilution of lowendotoxin rabbit complement (Accurate Chemicals, ACL-3051) which hadfirst been reconstituted in sterile water. The cell suspension wasincubated in complement for 30 minutes in a 37° C. water bath. Cellswere diluted in complete medium and washed twice by centrifugationbefore resuspending them in 5 ml of complete medium for counting.

[1448] 6. The cell concentration was adjusted to 3.3×10⁵ cells/ml incomplete medium containing 10 ng/ml IL-4 (Sigma) and 10 ng/mlrecombinant murine GM-CSF (Boehringer Mannhaim) and plated at 3 ml/wellin 6 well culture plates.

[1449] 7. Cultures were refed on day 5 by removal and replacement ofhalf the medium. This step was repeated as needed.

[1450] 8. At day 6 of culture, an aliquot of non-adherent and looselyadherent cells was collected and stained with antibody for N418 antigen(CD11c), a dendritic cell marker, using phycoerythrin (PE) labeledantibody. PE labeled antibody to an irrelevant antigen (TNP) served asthe negative control.

[1451] 9. Cultures were maintained until they were used for fusion withtumor cells.

[1452] 10. Staining and FACs analysis revealed approximately 75% of thepopulation to be CD11c positive.

[1453] Fusion of Dendritic cells with SEB Transfected B16 F10 MelanomaCells

[1454] One SEB positive clone (clone 11.2) and one vector containingclone (clone 7.5) were maintained in complete medium supplemented with50 mg/ml G418 and 0.5 mg/ml fungizone.

[1455] 1. Fusion of dendritic cells with the transtected B16F10 tumorcells, were carried out using preparations from seven and fourteen daycultures of dendritic cells.

[1456] 2. Fusion of the dendritic cells and tumor cells (either SEBtransfected or control vector) was carried out at a 3:1 ratio with PEG(1450 mwt) DMSO (Sigma P7306). Mock fusion consisted of mixing dendriticcells with SEB-transfected cells in the same ratio as used for fusionwithout PEG-DMSO. In a secpmd experiment, fusion of the dendritic cellsand the tumor cells was carried out at a 5:1 ratio.

[1457] 3. Four to 6 hours before fusion, tumor cells were cultured incomplete medium (see dendritic cell preparation) supplemented with 20%FCS and 1×OPI solution (oxaloacetate, pyruvate, and insulin, Sigma).Tumor cells and dendritic cells were mixed and washed three times withserum-free medium, After removing the medium, 1 ml of PEG was added tothe cell pellet while resuspending the cells by stirring for 2 min. Anadditional 10 ml of serum-free medium was added to the cell suspensionover the next three minutes with continued stirring. The cells werecentrifuged at 400×g for 5 min. and resuspended with complete medium 20%FCS. Cell suspensions were cultured in bulk cultures.

[1458] 4. In order to eliminate the melanoma cells remaining in the bulkculture, differential adherence subculturing was performed. Cultureswere “fractionated” into nonadherent (transfer of the upper half theculture medium with floating cells), loosely adherent (transfer of cellsresuspended by gentle pipeting), and adherent (cells requiringtrypsinization to recover). Cells transferred to fresh cultures wererefed with complete medium containing 20% FCS. Subculturing wasperformed every 2-3 days or as needed for approximately two weeks.

[1459] 5. Analysis of dendritic cell-melanoma cell fusions to detectCD11c positive cells was performed on cultures approximately 2 weeksafter fusion. Cells were plated at 2×10⁴cells/well in four well slidecultures. After three days in cultures, cells were stained with eitherantibody to N418 antigen (CD11c), a dendritic cell marker, usingphycoerythrin (PE) labeled antibody or PE labeled antibody to anirrelevant antigen (TNP) as the negative control. After washing withPBS, the cells were incubated for 30 minutes on ice in PBS with 10%FBSand Fc-Block (Pharmingen 01241D). Five μl/well of the appropriateantibody was added to the wells and the staining was carried out byincubating the slides for 30 minutes on ice. After three washes in PBS,cells were fixed with 2% paraformaldehyde. The remaining cells werefrozen for permanent storage.

EXAMPLE 26 Transfection of Hybrid DC/tc's with SAg DNA or RNA In Vivoand In Vitro

[1460] The methods for transfecting SAg-encoding nucleic acid into tumorcells disclosed in Examples 1 and 32 are used for transfection of DCs orDC/tc hybrids.

EXAMPLE 27

[1461] Preparation of DCs which have Phagocytosed SAg-Transfected TumorCell Lysates or Apoptotic Tumor Cells

[1462] PBMCs, DCs, macrophages, and T cells are prepared as follows. Inbrief, peripheral blood is obtained from normal donors in heparinizedsyringes and PBMCs are isolated by sedimentation over Ficoll-Hypaque(Amersham Pharmacia Biotech, Piscataway, N.J.). T cell-enriched and Tcell-depleted fractions are prepared by rosetting withneuraminidase-treated sheep red blood cells. Immature DCs are preparedfrom the T cell-depleted fraction by culturing cells in the presence ofGM-CSF and IL-4 for 7 d. 1,000 U/ml of GM-CSF (Immunex Corp., Seattle,Wash.) and 500-1,000 U/ml of IL-4 (Schering-Plough Corp., Kenilworth,N.J.) are added to the cultures on days 0, 2, and 4. To generate matureDCs, the cultures are transferred to fresh wells on day 7 and MCM isadded for an additional 3-4 d. At day 7, >95% of the cells areCD14−,CD83−, HLA-DR^(lo) DCs. On days 10-11, 80-100% of the cells are ofthe mature CD14−, CD83+, HLA-DRhi phenotype. FACSort® (Becton Dickinson,San Jose, Calif.) is used to generate highly pure populations ofimmature and mature DCs, based on their CD83− and CD83+ phenotypes,respectively. Macrophages are isolated from T cell-depleted fractions byplastic adherence for 1 h. After 24 h, cells are removed from the platesand placed in Teflon beakers for 3-9 d. T cells are further purifiedfrom the T cell-enriched fraction by removing contaminating monocytes,NK cells, and B cells.

EXAMPLE 28 Induction of Apoptotic Death and Phagocytosis of ApoptoticTumor Cells or SAg-Transfected Tumor Cells by DCs

[1463] Monocytes are infected with influenza virus in serum-free RPMI.These cells undergo viral-induced apoptotic death within 6-8 h. Celldeath is confirmed using the Early Apoptosis Detection kit (KayimaBiomedical Co., Seattle, Wash.). As previously described, cells arestained with Annexin V-FITC (Ann V) and propidium iodide (PI). Earlyapoptosis is defined by Ann V+/PI staining as determined by FACScan®(Becton Dickinson). Five to eight h after infection, monocytes firstexternalize PS on the outer leaflet of their cell membrane, as detectedwith Ann V. By 8-10 h, these cells are TUNEL (Tdt-mediated dUTP-biotinnick-end labeling) positive. It is not until 24-36 h that the majorityof the monocyte population included trypan blue into the cytoplasm, anindicator of secondary necrosis. HeLa cells are triggered to undergoapoptosis using a 60 UV lamp (Derma Control Inc.), calibrated to provide2 mJ/cm²/s.

[1464] Induction and Detection of Apoptosis

[1465] Monocytes are infected with influenza virus in serum-free RPMI.Cell death is assayed using the Early Apoptosis Detection kit (KayimaBiomedical). Briefly, cells are stained with Annexin V-FITC (Ann V) andpropidium iodide (P1). Early apoptosis is 14 defined by Ann V+/PI−staining as determined by FACScan (Becton Dickinson). Cells from the 293cell line are triggered to undergo apoptosis using a 60 UVB amp (DermaControl Inc.), calibrated to provide 2 mJcm⁻²s⁻¹.

[1466] Phagocytosis of Apoptotic Cells

[1467] Monocytes and HeLa cells are dyed red using PKH26-GL (SigmaBiosciences, St. Louis, Mo.), and induced to undergo apoptosis byinfluenza infection and UV irradiation, respectively. After 6-8 h,allowing time for the cells to undergo apoptosis, they are coculturedwith phagocytic cells that were dyed green using PKH67-GL (SigmaBiosciences), at a ratio of 1:1. Macrophages are used 3-6 d afterisolation from peripheral blood; immature DCs are used on days 6-7 ofculture; and mature DCs are used on days 10-11. Where direct comparisonof cells is needed, cells are prepared from the same donor on differentdays. In blocking experiments, the immature DCs are preincubated in thepresence of 50 mg/ml of various mAbs for 30 mm before the establishmentof cocultures. After 451 20 mm, FACScan® analysis is performed anddouble positive cells were enumerated.

[1468] Coculture of DCs with Apoptotic Cells

[1469] Monocytes from HLA-A2.1-donors are infected with live orheat-inactivated influenza virus. Live influenza virus (Spafas Inc.) isadded at a final concentration of 250 HAU ml-1 (MOI of 0.5) for 1 h at37° C. Virus is heat-inactivated by treatment for 30 min at 56° C.before use. After washing, cells are added to 24-well plates in varyingdoses. After 1 h, contaminating non-adherent cells are removed and freshmedia is added. Following a 10 h incubation at 37° C., 3.3×10³uninfected DCs and 10⁶ T cells are added to the wells.

[1470] Antigen Pulsing of DC

[1471] Day 7 DC are incubated with freeze-thawed tumor lysates at aratio of three tumor cell equivalent to one DC (i.e., 3:1) in CM. After18 hr of incubation, DC are harvested, irradiated with rad (Gamma Cell1000; Nordion, Kanata, Canada), washed twice in Hank's balanced saltsolution (GIBCO), and in Hank's balanced salt solution.

EXAMPLE 29

[1472] Treatment of Tumor Bearing Animals with SAg-Transfected orSAg-Expressing DCs, Accessory Cells or S/D/t Cells: VaccinationProtocols and Treatment of Established Tumor

[1473] Immunotherapy

[1474] C57BL/6 mice are immunized once with irradiated, S/D/t cells(2×10⁶ cells/mouse) 10-14 d post-immunization mice are challenged with2×10⁷ live tumor cell subcutaneously in the scapular region. Mice aremonitored on a regular basis for tumor growth and size. Mice with tumorsizes >3.5 cm were killed. All survivors were killed 40 dpost-challenge.

[1475] P10.9-B 16 Melanoma Model

[1476] Mice are injected intra-footpad with 2×10⁵ F10.9 cells. Legs areamputated when the local tumor in the footpad is 7-8 mm in diameter.Post-amputation mortality is less than 5%. 2 d post-amputation mice areimmunized intraperitoneally with S/D/t cells followed by weeklyvaccinations twice, for a total of three vaccinations. Mice are killedbased on the metastatic death in the non-immunized or control groups(28-32 d post-amputation). Metastatic loads are assayed by weighing thelungs.

[1477] S/D/t cells: In Vivo Immunization and Tumor Challenge

[1478] B6 or BALB/c mice are immunized s.c. in the right flank with 10⁶MCA-207 or 10⁶ S/D/t cells, respectively, twice at 7-day intervals. Micethen are rechallenged 7 days after the last immunization with a lethaldose of 10⁵ MCA-207 (for B6 mice) or 3×10⁵ MT-901 (for BALB/c mice)viable tumor cells by s.c. injections into the left flank. The size ofthe tumors is assessed in a blinded, coded fashion twice weekly andrecorded as tumor area (in square mm) by measuring the largestperpendicular diameters with calipers. Data are reported as the averagetumor area SEM (five or more mice per group).

[1479] Vaccination Protocol

[1480] B6 mice are s.c. immunized twice in a 2-wk interval with 10⁶irradiated (15,000 rad) B16, B16 mixed with DCs (1/1: unfractionatedcells from overnight culture), or S/D/t cells or recombinant formalinfixed bacteria (10⁶-10⁸). Ten days following the final immunization,each group of mice is injected s.c. with varying doses (10⁴, 10⁵, or 10⁶cells/mouse) of viable B16. Tumor growth and survival time of each groupof mice are recorded. The size of the tumor in each mouse is measured intwo perpendicular dimensions with a Vernier caliper twice weekly aftertumor challenge. Tumor incidence is considered positive when the averagediameters of the tumor exceeded 3 mm.

[1481] In Vivo Immunization for Treatment of Pulmonary Metastases

[1482] B6 or BALB/c mice receive 1.5×10⁵ MCA-207 or 2×10⁵ MT-901 viabletumor cells, respectively, i.v. in the lateral tail vein to establishpulmonary metastases, as described. The mice then are immunized s.c.with, respectively, 10⁶ MCA-207 tumor lysate-pulsed S/D/t cells threetimes on days 3, 7, and 11 or 10⁶ MT-901 tumor lysate-pulsed S/D/t cellstwice on days 3 and 7 after tumor injection and are killed on days 14and 17, respectively. Pulmonary metastases are enumerated on day 15(MCA-207) or 14 (MT-901). Data are reported as the mean number ofmetastases±SEM (five or more mice per group).

[1483] In Vitro Activation of LN T Cells

[1484] B6 mice are immunized s.c. twice in a 2-wk interval on the flankswith 2×10⁶ (10⁶/side) irradiated (15,000 rad) tumor, S/D/t cellpreparation, or tumor mixed with DCs (1/1) suspended in 0.1 ml of HBSS.One week after the final immunization, inguinal LNs from each group ofmice are harvested. LN cells from each group of mice are activated andexpended in culture using anti-CD3 plus IL-2. In brief, LN cells(3-4×10⁶ cells/well) are activated on 24-well plates coated withanti-CD3 mAb (145-2C11) and incubated at 37° C. for 2 days.Alternatively, S/D/t cells (10⁴-10⁵/well 5 g) or recombinant bacteria(10⁶-10⁸/well) are incubated with the LN cells for 2 days and optionallywith low dose IL-2 for an additional 2 days. The activated cells aresuspended at 1-2×10⁵ cells/ml in CM containing IL-2 (4 U/ml) andincubated in gas-permeable culture bags (Baxter Healthcare, Deerfield,Ill.) for an additional 3 days. The derived LN T cells are harvested andused as effector cells for adoptive immunotherapy.

[1485] Adoptive Immunotherapy Models

[1486] For therapy of B 16 pulmonary metastases. B6 mice are injectedi.v. with 10⁵ live B16 tumor cells in 1 ml of PBS to initiate pulmonarymetastases. Three days after tumor inoculation, mice are randomlydivided into several groups to receive treatments by i.v. injection of5×10⁷ cultured LN T cells suspended in 1 ml of PBS. On day 21 aftertumor inoculation, mice from each group are killed, and lungs areinsufflated with Fekete's solution. Lung metastases are counted. In someexperiments. tumor-bearing mice are i.p. administered IL-2 (15,000 U.twice/day for 5 days) following the adoptive transfer of cultured LN Tcells. For therapy of FBL-3 tumor. B6 mice are inoculated i.p. with5×10⁶ viable FBL-3 tumor cells on day 0. By day 5, the tumor isdisseminated, and mice are treated with cyclophosphamide (CY) at a doseof 180 mg/kg followed in 6 h by i.p. injection of cultured LN T cells(5×10⁷ cells/mouse) suspended in 0.5 ml of PBS. The tumor growth and thesurvival time of each group of mice are monitored and recorded on aregular basis.

[1487] Induction of Anti-Tumor Activity by FC/MUC1.

[1488] Groups of 1 mice are immunized twice at 14-day intervals bysubcutaneous injection of 3×10⁵ DCs (0) or S/D/t cells represented byFC/MUC1cells. PBS is injected as a control (0). After 14 days, mice arechallenged subcutaneously with 2.5×10⁵ MC38/MUC1 cells. Tumors of 3 mmin diameter are scored as positive.

[1489] Immunization with FC/MUC1 for Prevention and Treatment ofPulmonary Metastases

[1490] Groups of 10 mice are injected twice with S/D/t cells representedby FC/MUC1cells or PBS and then challenged after 14 days withintravenous administration of 10⁶ MC38/MUC1 cells. The mice are killed28 days after challenge. Pulmonary metastases are enumerated afterstaining the lungs with India ink. Groups of 10 mice are injectedintravenously with 10⁶ MC38/MUC1 or MC38 cells. The mice are immunizedwith 10⁶ S/D/t cells representing FC/MUC1 cells or FC/MC38 at 4 and 18days after tumor challenge and then killed after an additional 10 days.Pulmonary metastases are enumerated for each mouse.

[1491] Protection Assays

[1492] C57BL/6 mice are immunized with the indicated antigen-geneconstruct. Animals are challenged with tumors and evaluated for tumorsurvival as described. Briefly, 7 days after the final immunization (day0), immunized animals are challenged by intradermal injection in themid-flanks bilaterally with melanoma cells (2×10⁴) at two times the doselethal to 50% of the animals tested (LD50). Survival is recorded as thepercentage of surviving animals. Melanoma cells for injection are washedthree times in PBS. Injected cells were greater than 95% viable bytrypan blue exclusion. All experiments include five mice per group andwere repeated at least three times. Mice that became moribund werekilled according to animal care guidelines.

EXAMPLE 30 DNA or RNA from SAg Transfected Tumor Cells, SAg TransfectedDCs and SAg Transfected DC/tc Hybrids for In Vivo Vaccination andTransfection of Naive DCs to Produce a DC Expressing SAgs and TumorAssociated Antigens

[1493] Plasmid DNA Vector

[1494] 1. Genes from SAg Transfected Tumor Cells, SAg transfected DCsand S/D/t cells are cloned by PCR to contain a partial or entire codingregion. In most cases, it is desirable to not include any sequence 5′ tothe ATG or 3′ to the termination codon. PCR primers are designed tocontain a restriction site, such as BgIII or BamHI.

[1495] 2. The PCR fragments are separated from unreacted oligomers andtemplate and then the fragment is cut with an excess of BgIII for atleast 5 h. The DNA is Phenol extracted and ethanol-precipitated. Thepurified cut fragment is resuspended in TE, pH 8.0 and ligated. toBgIII-digested V1J, which has been gel-purified and dephosphorylatedwith calf intestinal alkaline phosphatase (CLAP), phenol-extracted,ethanol-precipitated, and resuspended in TB, pH 8.0. A 6:1 molar ratioof insert:vector in the ligation reaction is used.

[1496] 3. Competent E. coli cells (e.g., DH5, DH5a) are transformed withthe ligation reaction, plated on L-ampicillin plates and grown overnightat 37° C. Colonies are screened by hybridization of plate lifts tokinase-labeled PCR primer. Several hybridization-positive colonies areselected and grown in overnight cultures for miniprep purification.

[1497] 4. Miniprep DNAs, are prepared and cut with the appropriaterestriction enzymes to determine correct orientation of the gene in thevector. At least three DNAs with the gene in the correct orientation areselected to confirm by sequencing across the ligation junctions.Sequencing primers are designed from the vector sequence. Each primer is30-50 bp from. the restriction site (BgIII in the example), so that10-20 bases within the vector can be read as well as 150-200 baseswithin the gene. This amount of sequence verifies orientation and give areasonable estimate of the quality of the PCR-generated gene.

[1498] 5. DNA preparations that have been sequence-verified 1/1000 inTB, pH 8.0, are diluted and use to retransform competent E. coli. Threeisolated colonies from the transformation plates are grown overnight at37° C., and used to make a −70° C. cell stock by adding 0.8 ml freshovernight growth to 0.2 ml sterile 80% (v/v) glycerol, mixing well, andfreezing on dry ice. The −70° C. stocks are used to isolate plasmid DNAfrom remaining cells by miniprep procedures. Miniprep DNA is cut againwith the appropriate restriction enzymes, and visualized on a gel toverify the construct. All subsequent growth of cells for plasmidproduction are made from the −70° C. frozen stock.

[1499] All constructs are tested in vitro to validate their ability toexpress the desired gene product. Plasmids purified by column (Wizardpreps, Promega, Madison, Wis.) or by cesium chloride banding are used totransfect tissue-culture cells transiently. Protein expression isdetected by immunoblot. This check not only verifies expression but canvalidate the size and immunoreactivity of the gene product.

[1500] Characterization of Plasmid DNA Vectors

[1501] All constructs are tested in vitro to validate their ability toexpress the desired gene product. Plasmids purified by column (Wizardpreps, Promega, Madison, Wis.) or by cesium chloride banding are used totransfect tissue-culture cells transiently. Protein expression isdetected by immunoblot. This check not only verifies expression but canvalidate the size and immunoreactivity of the gene product.

[1502] Cell Growth and Transfection

[1503] 1. DC /tumor cell hybrids, at 0.8-1.5×10⁶ cells/100 mm plate inDulbecco's Modified Eagle's Medium (DMEM) supplemented with 10%heat-inactivated fetal bovine serum, 20 mM HEPES, 4 mM L-glutamine, and100 mg/ml each of penicillin and streptomycin, and incubate at 37° C. in5% CO2 for 18 h.

[1504] 2. The construct to be tested is cotransfected with 10 mg/plateand 10 g of V1J-CAT using a calcium phosphate procedure or other methodsgiven in Example 1.

[1505] 3. Five hours after transfection, the cells are shocked in 15%(v/v) glycerol in PBS, pH 7.2, for 2.5 mm.

[1506] 4. Cultures are harvested 72 h after transfection by washing theplates twice with 10 ml of cold PBS, pH 7.2, then adding 5 ml of coldTEN buffer and scraping.

[1507] 5. Pellet cells and use immediately or store at −70° C. forsubsequent analysis.

[1508] Immunoblot Analysis

[1509] 1. Cell pellets are lysed in Single Detergent Lysis Buffer, andsonicate on ice (2-15 s bursts) to reduce viscosity.

[1510] 2. Cell debris is removed by sedimentation and determine solubleprotein concentrations of the supernatants by the Bradford method.

[1511] 3. Equal loadings of soluble cell protein per lane are run onSDS-polyacrylamide gel and transfer the proteins to Immobilon P(Millipore, Bedford, Mass.) membrane.

[1512] 4. Western blots are incubated overnight with an appropriatedilution of the antibody to the gene product being tested, followed by a1.5-h reaction with a 1:1000 dilution of peroxidase-conjugated secondaryantibody. Develop blots using the ECL kit (Amersham, Arlington Heights,Ill.).

[1513] Large-Scale DNA Preparations

[1514] 1. Expression vectors are grown in E. coli strain DH5 withvigorous aeration in 500 ml growth medium/1-L shake flask. V1Jconstructs are grown overnight to saturation.

[1515] 2. Cells are harvested and lysed by a modification of thealkaline SDS procedure. The modification consists of increasing thevolumes threefold for cell lysis and DNA extraction.

[1516] 3. DNA is purified by double banding on CsC1/ethidium bromidegradients.

[1517] 4. The ethidium bromide is removed by 1-butanol extraction, andthe resulting DNA is extracted with phenol/chloroform and precipitatedwith ethanol.

[1518] 5. DNA in TE for transfections is resuspended and in 0.9% NaClfor injection into mice.

[1519] 6. The concentration and purity of each DNA preparation isdetermined by A 260/280 readings. The 260/280 ratios are >1.8.

[1520] 7. DNA is stored in small aliquots at −20° C.

EXAMPLE 31 DNA Immunization In Vivo

[1521] 1. Animals are housed in an American Association for theAccreditation of Laboratory Animal Care (AAALAC) accredited facility orother national facility and cared for in accordance with the Guide forthe Care and Use of Laboratory Animals. Prior to bleeding, oradministration of anesthetic or inoculation, animals are in goodphysical condition and free from stress.

[1522] 2. For administration of DNA vaccines, animals are anesthetizedby ip injection of a solution containing ketamine and xylazine (50 and20 mg/g body wt, respectively) in a total volume of 0.3 ml of saline.Alternatively, transiently immobilize mice for a sufficient period oftime to administer an im injection by allowing inhalation of metophane.Larger animals, such as ferrets or nonhuman primates, are anesthetizedusing ketamine (30 mg/kg)/xylazine (2 mg/kg)/atropine (1 mg/kg) orketamine (10 mg/kg), respectively.

[1523] 3. Fully anesthetized animals are prepared for injection byflooding and swabbing the injection site with ethanol (70%). Thisprovides sterilization and, for small animals, such as mice, facilitatesvisualization of the muscle groups. To visualize small muscles further,fur around the injection site is shaved followed by ethanol swabbing, ora short incision can be made to permit direct observation of the muscle.In the latter case, the incision is sutured after inoculation.

[1524] 4. DNA vaccines are administered in saline solution alone ortogether with a facilitator that induces muscle generation orregeneration. Facilitators are used in animals that may not necessarilybe used in humans. For mice, volumes of up to about 50 mL are injectedinto each quadriceps muscle using a disposable insulin syringe equippedwith a 27-gauge needle and having a capacity of 0.3 ml.

[1525] 5. DNA vaccines are also administered using particle bombardmenttechnology. Plasmid DNA is coated onto gold beads and propelled directlyinto tissue. Genetic immunization is accomplished by biolisticbombardment using methods similar to those recently described. Briefly,DNA-coated gold particles are prepared by combining 50 mg of 0.95 umgold beads and 100 l of 0.1 M spermidine and sonicating for 5 s. PlasmidDNA (100 mg) and CaCl (200 ml) are added sequentially to the beadsspinning in a vortex; mixer. This mixture is allowed to precipitate atroom temperature for 5-10 mm. The bead preparation is then centrifuged(10,000 r.p.m. for 30 s) and washed 3 times in cold ethanol beforeresuspension in 7 ml of ethanol to give a final concentration of 7 mggold per milliliter. The solution is then loaded into Tefzel tubing(Agracetus, Middleton, Wis.) and allowed to settle for 5 mm. The ethanolis removed and the beads are attached to the side of the tubing byrotation at 20 r.p.m. for 30 s and N₂ dried. The dried tubing lined withbeads is then cut into 0.5-inch sections and stored for use withdesiccant in parafilm-sealed vials. Animals are vaccinated by deliveryof two shots (each shot consisted of 0.5 m4j gold beads in 0.5 inch oftubing) to the shaved abdominal region using the Accell gene deliverydevice (Agracetus) at a discharge pressure of 400 p.s:i. This deliversapproximately 1.00 mg/DNA per shot. Animals are immunized with variousplasmids In some instances, particles,are coated with the pGREENLANTERN-1 plasmid (Gibco BRL, Gaithersburg, Md.), which contains the“humanized” reporter gene encoding GFP from the Aequorecia victoriajellyfish. This gene encodes a naturally fluorescent protein requiringno substrates for visualization.

[1526] Formulation of DNA Vaccine

[1527] Saline is the preferred solvent. However, plasmid DNA may also beadministered in various other buffer formulations and cationic lipidformulations. Facilitators include anesthetics, such as bupivacaine, andtoxins, which are used in conjunction with DNA vaccines. Conventionaldelivery vehicles are used which facilitate internalization of DNA bycells, protect DNA from digestion by extracellular nucleases, or effecta slow release of DNA; adjuvants are coadministered to provide anadditional stimulus for the immune system.

[1528] Dosage and Injection Regimen

[1529] DNA vaccines are effective across a broad dosage range.Protective efficacy is achieved with submicrogram amounts of DNA. Withrespect to humoral immune responses against HA, there is a directcorrelation between magnitude of antibody responses and dose of DNAbetween 10 ng and at least 100 g. However, perhaps owing to viscosity ofthe solution and/or distribution of the inoculum in the muscle,administration of DNA at concentrations in excess of 2-4 mg/ml resultsin decreased immunogenicity with some antigens. Therefore, in mice,doses in excess of 200 g are not practical by im injection. The numberof injections also directly correlates with magnitude of immuneresponses (up to at least three). For the influenza model in mice, wehave found that three injections given at 3-wk intervals yield optimalprotection. It is likely, however, that dosing and regimen will need tobe optimized for each gene and challenge model.

[1530] Site of Injection

[1531] Injection of plasmid DNA into muscle cells is far superior toother cell types in their capacity to internalize DNA and/or expressreporter proteins in vivo. However, immune responses also have beengenerated after id and iv routes of DNA injection. In addition, particlebombardment of DNA results in the transfection of dermal and epidermalcells leading to the generation of immune responses. The relativeeffectiveness of these different routes of delivery has yet to be testedrigorously. However, direct im injection generates a protective immuneresponses at doses (100 ng to 1 g) and is preferred in the range used byparticle bombardment.

EXAMPLE 32 Pulsing DCs with RNA from SAg Producing Bacteria or S/D/tCells

[1532] Total RNA is isolated from SAg producing bacteria or S/D/t cellsby standard methods. Pulsing DCs with RNA from SAg producing bacteria,S/D/t cells or SAg transfected tumor cells is performed in serum-freeOpti-MEM medium (GIBCO BRL) for tumor extracts with the followingmodification RNA (25 gin 250 l Opti-MEM medium) and DOTAP (50 g in 250 lOpti-MEM medium) are mixed in 12×75 mm polystyrene tubes at roomtemperature for 20 mm. The complex is added to the DCs (25×10⁶ cells/ml)and incubated 37° C. in a water bath with occasional agitation for 25mm. The cells are washed twice and resuspended in PBS (10⁵ RNA pulsedDCs in 500 l PBS/mouse) for intraperitoneal immunizations. PBS, B16extract from 10⁵ cells in PBS, or DCs prepared as described above areinjected intraperitoneally in a volume of 500 l.

EXAMPLE 33 PolyA-Cellular RNA from S/D/t Cells or DCs Transfected withSAg

[1533] Preparation and Immunization Protocols

[1534] Total RNA is isolated from actively S/D/t cells given above asfollows. Briefly, 10⁷ cells are lysed in 1 ml of guanidiniumisothiocyanate (CT) buffer (4 M guanidinium isothiocyanate, 25 mM sodiumcitrate, pH 7.0; 0.5% sarcosyl, 20 mM EDTA, 0.1M 2-mercaptoethanol).Samples are vortexed followed by sequential addition of 100 l 3M sodiumacetate, 1 ml water saturated phenol and 200 l chloroform/isoamylalcohol (49:1). Suspensions are vortexed and placed on ice for 15 mm.The tubes are centrifuged at 10,000 g, 4° C. for 20 min and thesupernatant is carefully transferred to a fresh tube. An equal volume ofisopropanol is added and the samples are placed at −20° C. for at least1 h. RNA is pelleted by centrifugation as above. The pellet isresuspended in 300 l GT buffer which is then transferred to amicrocentrifuge tube. RNA is re-precipitated by adding an equal volumeof isopropanol and placing the tube at −20° C. for at least 1 h. Tubesare microcentrifuged at high speed at 4° C. for 20 mm. Supernatants aredecanted and pellets are washed once with 70% ethanol. Pellets areallowed to dry at RT and then resuspended in TB (10 mM Tris-HCl, 1 mMEDTA, pH 7.4). Possible contaminating DNA is removed by incubating RNAin 10 mM MgCl2, 1 mM DTT and 50 U/ml RNase free DNase(Boehringer-Mannheim, Indianapolis, Ind.) for 15 min at 37° C. Thesolution is adjusted to 10 mM Tris, 10 mM EDTA, 0.5% SDS and 1 mg/mlPronase (Bochringer-Mannheim) followed by incubation at 37° C. for 30mm. Samples are extracted once with phenol-chloroform and once withchloroform, and RNA was then re-precipitated in isopropanol at −20° C.After centrifugation the pellets are washed with 70% ethanol, air dried,and resuspended in sterile water. Total RNA is quantitated by measuringOD at 260 and 280 nm. OD 260/ 280 ratios are typically 1.65-2.0. RNA isstored at −70° C. PolyA+ RNA is either isolated from total RNA usingOligotex (Qiagen, Chatsworth, Calif.) or directly from tissue culturecells using the Messenger RNA Isolation kit (Stratagene, La Jolla,Calif.) as per manufacturer's protocols.

[1535] Production of In Vitro Transcribed RNA

[1536] The 1.9-kb EcoRI fragment containing the coding region and 3′un-translated region is cloned into the EcoRI site of pGEM4Z (Promega,Madison, Wis.). Clones containing the insert in both the sense andanti-sense orientations are isolated and large scale plasmidpreparations are made using Maxi Prep Kits (Qiagen). Plasmids arelinearized with BamHI for use as templates for in vitro transcription.Transcription is carried out at 37° C. for 34 h using the 5P6 MEGAscriptIn vitro Transcription Kit (Ambion, Austin, Tex.) per manufacturer'sprotocol and adjusting the GTP concentration to 1.5 mM and including 6mM m7G(5′)ppp(5′)G cap analogue (Ambion). Template DNA is digested withRNase free DNase I and RNA is recovered by phenol/chloroform andchloroform extraction followed by isopropanol precipitation. RNA ispelleted by microcentrifugation and the pellet is washed once with 70%ethanol. The pellet is air-dried and resuspended in sterile water. RNAis incubated for 30 mm at 30° C. in 20 mM Tris-HCl, pH 7.0, 50 mM KCl,0.7 mM MnCl2, 0.2 mM EDTA, 100 mg/ml acetylated BSA, 10% glycerol, 1 mMATP and 5,000 U/ml yeast poly(A) polymerase (United States Biochemical,Cleveland, Ohio). The capped, polyadenylated RNA is recovered byphenol/chloroform and chloroform extraction followed by isopropanolprecipitation. RNA is pelleted by microcentrifugation and the pellet iswashed once with 70% ethanol. The pellet is air-dried and resuspended insterile water. RNA is quantitated by measuring OD at 260 and 280 nm andstored at −70° C.

[1537] Pulsing of Antigen-Presenting Cells, Accessory Cells DCs, TumorCells or DC/Tumor Cell Hybrids with RNA Derived from S/D/t Cells

[1538] Pulsing of cells with RNA is routinely performed in serum-freeOpti-MEM medium (GIBCO BRL). Cells are washed twice in Opti-MEM medium.Cells are resuspended in Opti-MEM medium at 25×10⁶ cells/nil and addedto 15 ml polypropylene tubes (Falcon). The cationic lipid, DOTAP,(Boehringer Mannheim) is used to deliver RNA into cells. RNA (in 250-500l Opti-MEM medium) and DOTAP (in 250-500 p.1 Opti-MEM medium) are mixedin 12×75-mm polystyrene tubes at room temperature (RT) for 20 mm. Theamount of polyA+ RNA or IVT RNA used is 5 g and the amount of total RNAused is 25 g. The RNA to DOTAP ratio is 1:2. The complex is added to theAPC (2-5×10⁶ cells) in a total volume of 2 ml and incubated at 37° C. ina water-bath with occasional agitation for 2-4 h.

EXAMPLE 34 In Vivo Immunization with RNA Derived from “S/D/t Cells” orSAg-Transfected Tumor Cells

[1539] Preparation of mRNA for Transfection

[1540] DNA is linearized downstream of the poly A tail with a 5-foldexcess of PstI. The linearized DNA is then purified with twophenol/chloroform extractions, followed by two chloroform extractions.DNA is then precipitated with NaOAc (0.3M) arid 2 volumes of EtOH. Thepellet is resuspended at about 1 mg/ml in DEP-treated deionized water.

[1541] A transcription buffer is prepared, comprising 400 mM Tris. HCl(pH 8.0), 80 mM MgCl2, 50 mM DTT, and 40 mM spermidine. The followingmaterials are added in order to one volume of DEP-treated water at roomtemperature: 1 volume T7 transcription buffer; rATP, rCTP, and rUTP to 1mM concentration; rGTP to 0.5 mM concentration; 7 g(5′)ppp(5′)G capanalog (New England Biolabs, Beverly, Mass.) to 0.5 mM concentration;the linearized DNA template to 0.5 mg/ml concentration; RNAsin (Promega,Madison, Wis.) to 2000 U/ml concentration; and T7 RNA polymerase (N.E.Biolabs) to 4000 U/ml concentration.

[1542] This mixture is incubated for 1 hour at 37° C. The successfultranscription reaction is indicated by increasing cloudiness of thereaction mixture.

[1543] Following generation of the mRNA, 2U RQ1 DNAse (Promega) permicrogram of DNA template used is added and was permitted to digest thetemplate for 15 minutes. Then, the RNA is extracted twice withchloroform/phenol and twice with chloroform. The supernatant isprecipitated with 0.3M NaOAc in 2 volumes of EtOH, and the pellet isresuspended in 100 mu 1 DEP-treated deionized water per 500 ltranscription product. This solution is passed over an RNAse-freeSephadex G50 column (Boehringer Mannheim #100 411). The resultant mRNAis sufficiently pure to be used in transfection of vertebrates in vivo.

[1544] mRNA Vaccination In Vivo

[1545] A liposomal formulation containing mRNA coding for the SAg/tumorassociated antigen protein prepared and is inserted into the plasmidpXBG in A volume of 200 l of a formulation is prepared containing 200mg/ml of S/D/t cell-derived mRNA and 500 mg/ml 1:1 DOTAP/PE in 10%sucrose is injected-into the tail vein of mice 3 times in one day. Atabout 12 to 14 h after the last injection, a segment of muscle isremoved from the injection site, and prepared as a cell lysate accordingto Example 7. The S/D/t cell-derived specific protein is identified inthe lysate.

[1546] Severe combined immunodeficient (SCID) mice (Molecular BiologyInstitute, (MBI), La Jolla, Calif.) were reconstituted with adult humanperipheral blood lymphocytes by injection into the peritoneal cavityaccording to the method of Mosier (Mosier et al., Nature 335:256(1988)). The mice were maintained in a P3 level animal containmentfacility in sealed glove boxes. mRNA coding for the S/D/t cell-derivedproteins is prepared by obtaining the S/D/t cell gene in the form of aplasmid removing the gene from the plasmid; inserting the gene into thepXBG plasmid for transcription; and purifying the transcription productS/D/t cell-derived mRNA. The S/D/t cells mRNA is then incorporated intoa formulation and 200 l tail vein injections of a 10% sucrose solutioncontaining 200 mg/ml S/D/t cell RNA and 500mg/ml 1:1 DOTAP:DOPE (inRNA/liposome complex form) were performed daily on experimental animals,while control animals were likewise injected with RNA/liposome complexescontaining 200 mg/ml yeast tRNA and 500 mg/ml 1:1 DOTAP/DOPE liposomes.At 2, 4 and 8 weeks post injection, biopsy specimens are obtained frominjected lymphoid organs and prepared for immunohistochemistry.

[1547] A volume of 200 l-of the formulation, containing 200 mg/ml mRNAfrom S/D/t cells, and 500mg/ml 1:1 DOTAP:DOPE in 10% sucrose is injectedinto the tail vein of the human stem cell-containing SCID mice 3 timesin one day. Following immunization, the mice are challenged by tumorinoculation.

[1548] The full-length sequence for the cDNA of the S/D/t-derived geneis obtained and ligated to BgIII linkers and then digested with BgIII.The modified fragment is inserted into the BgIII site of PXBG.S/D/t-derived protein is transcribed and purified mRNA is incorporatedinto a formulation. Balb 3T3 mice are injected directly in the tail veinwith 200 l of this formulation, containing 200 mg/ml of S/D/t-derivedmRNA, and 500 mg/ml DOTAP in 10% sucrose.

EXAMPLE 35 Preparation of “String of Beads” Tumor Antigens forTransfection of SAg-Transfected DCs, Other Accessory Cells, or TumorCells

[1549] Generation of rAd

[1550] All cell lines were maintained in Iscove's modified Dulbecco'smedium (IMDM) (Scromed, Berlin) supplemented with 4% fetal calf serum(FlyClone), penicillin (110 international units/ml; Brocades Pharma,Leiderdorp, The Netherlands) and 2-mercaptoethanol (20 mM) at 37° C. ina 5% CO2 atmosphere. The adenoviral vector construction adapter plasmidpMad5 is derived from plasmid pMLP10 as follows. pMLP10-lin isconstructed by insertion of a synthetic DNA fragment with unique sitesfor the restriction endonucleases MluI, SplI, SnaBI, SpeI, AsulI, andMunI into the HindIII site of pMLP10. Subsequently, the adenovirus BgIIIfragment spanning nucleotides 3328 8914 of the AdS genome is insertedinto the MunI site of pMLP-lin. Finally, the SalI-BamHI fragment isdeleted to inactivate the tetracycline resistance gene, resulting inplasmid pMad5. A mini-gene cassette vector, pMad5-0. is generated byligation of the annealed and phosphorylated double-strandedoligonucleotides 1a/b and 2a/b into the MluI and SpeI sites of pMad5.This cloning step leads to elimination of the original MluI and SpeIsites and to creation of a small ORF, which essentially consists of astart codon, the sequence SEOKLISEEDLNN, a human c-Myc-derived sequence,which is recognized by mAb 9E10 and a stop codon. A small “stuffer”sequence. flanked by newly generated MluI and SpeI sites, is presentbetween the start codon and the c-Myc sequence.

[1551] pMad5-1 and -2, each of which harbor a multi-epitope encodingminigene are constructed by unidirectional cloning of the followingdouble-stranded oligonucleotides into pMad5-0, which had been cleavedwith MluI and SpeI. pMad5-I. After each cloning step, the sequence ofthe inserts is verified by DNA sequencing. Expression of these minigenesis driven by the Ad5 major late promoter, which in this configuration islinked to the AdS immediate early enhancer, resulting in immediate earlyexpression of the minigenes.

[1552] rAds are generated through in vivo homologous recombination inthe Ad5E 1-transformed helper cell line 911 between plasmid pJMI7.containing the sequence of the AdS mutant d1309, and either of theplasmids pMad5-1 or pMad5-2. 911 cells are transfected with 10 g ofplasmid pJMI7 in combination with 10 g of either pMad5-1 or pMad5-2. TherAds are plaque-purified three times, after which the clonal rAds arepropagated in 911 cells, purified by double cesium chloride densitygradient centrifugation and extensively dialyzed. The presence ofreplication-competent adenoviruses is routinely checked by infection ofHep-G2 cells. The viral stocks were stored in aliquots with 10% glycerolat −80° C. and titered by plaque assay using 911 cells.

[1553] Further Transfection of SAg-Transfected DCs, Accessory Cells, orTumor Cells

[1554] In short, 100 ng of plasmid DNA encoding Ad5LI, HPV 16 E7, murinep53 or the influenza-matrix protein are transfected into 10⁴SAg-transfected DCs, accessory cells or tumor cells. The transfectedcells are incubated in 100 ml of IMDM containing 8% fetal calf serum for48 h at 37° C., after which 1500-500 CTL.??? in 25 ml of IMDM containing50 Cetus units (=300 international units) of recombinant interleukin-2(Cetus) are added. After 24 h, the supernatant is collected, and itstumor necrosis factor (TNF) content is determined by measuring itscytotoxic effect on WEHI- 164 clone 13 cells.

EXAMPLE 36 Production of Exosomes from DCs Expressing SAg and TumorAssociated Antigens and Normal Hepatocytes

[1555] Exosome Isolation

[1556] SAgs or tumor associated antigens are transfected into tumorcells, DCs, or DC/tc hybrids by methods disclosed herein. TheSAg-encoding nucleic acid is provided with sorting sequences which routethe translated protein to the endoplasmic reticulum and thereupon tosecretory vesicles or exosomes. Alternatively, tumor cells, DCs or DC/tcare incubated 18-20 hours with tumor peptides or SAgs. DCs supernatantsare harvested, centrifuged (at 4° C.) at 300 g for 20 mm and then at10,000 g for 30 min (to eliminate cell debris). Exosomes are thenpelleted at 100,000 g for one hour, and washed once in a large volume ofPBS (over 100-fold the final volume of resuspension of the exosomes).The protein concentrations in exosome preparations is measured byBradford assay (BioRad). The slightly acidic pH transiently induced bythe acid peptide elution increases the amounts of exosomes produced byDCs. Three to five g of exosomes are routinely obtained from 5-10×10⁵DCs in 18-20 hours. Exosomes containing LDL, oxyLDL, apolipoproteins,LDL receptors and oxyLDL receptors are obtained from normal hepatocytesby a method similar to that described above for dendritic cells andsickled erythrocytes as in Example 6.

[1557] Mice and Tumor Cell Lines for Exosome Trials

[1558] DBA/2J (H-2d) and BALB/c (H-2d) female mice 6-8 weeks of age areraised in pathogen-free conditions. P815 (H-2d) is a methylcholanthreneinduced mastocytoma, syngeneic with DBA/2. TS/A (H-2d) is aspontaneously-arising undifferentiated mammary adenocarcinoma, syngeneicwith BALB/c. All tumor cell lines are maintained in RPMI 1 640supplemented with 10% endotoxin-free fetal calf serum (Gibco BRL), 2mML-Glutamine, 100 U/ml penicillin, 100 mg/ml streptomycin, essentialamino acids and pyruvate.

[1559] Experimental Mouse Models for Exosome Trials

[1560] Twice the minimal tumorigenic dose of tumor cells (5×10⁵ P815,10⁵ TS/A) is inoculated intradermally in the upper right flank of DBA/2and BALB/c mice, respectively. Animals with established tumors at days3-4 for TS/A, or days 8-10 for P815, are immunized with a singleintradermal injection of 3-5 g of exosomes per mouse in the loweripsilateral flank. The tumor size is monitored biweekly and mice aresacrificed when bearing ulcerated or huge tumor burdens. All experimentsare performed two to three times using individual treatment groups offive mice per group.

EXAMPLE 37 Bacterial Constructs for the Expression of SAgs Linked toGalactosylceramides, α-Gal Epitope. Peptidoglycans, Lipopolysaccharidesand β1,3-Glucans

[1561] Nucleic acids encoding SAgs may be transfected into bacteriawhich naturally synthesize and express fundamental recognition units forinnate immunity. Some of these moieties such as monogalactosylceramidesand α-gal actosylceramides are potent immunogens and induce anti-tumoractivity. The addition of the SAg and a dominant tumor associatedepitope coexpressed with these natural bacterial constructs andadministered to a tumor bearing host would promote a potent tumorspecific response. The system described uses S. carnosus as a modelbacterial system to express a SAg peptide and dominant tumor epitope.

[1562] Expression Vectors for Surface Display

[1563] The shuttle vector constructed pSPPmABPXM consists of thefollowing parts: (i) the origin of replication for E. coli and the13-lactamase gene giving ampicillin resistance for transformed E. colicells; (ii) the origin of replication for phage f1; (iii) the origin ofreplication from S. aureus and the chloramphenicol acetyltransferasegene for staphylococcus expression; (iv) the promoter, signal sequence,and propeptide sequences from the S. hyicus lipase gene construct,optimized for expression in S. carnosus; (v) a multicloning sitecontaining three unique recognition sites for restriction endonucleases;(vi) a gene fragment encoding a serum ABP from streptococcal protein G;and (vii) gene fragments encoding the cell wall-anchoring regions X andM from staphylococcal protein A.

[1564] As a model system, the surface display of SAg staphylococcalenterotoxin B. substitutes for place the 80 amino acid malaria peptideM3 from falciparum blood stage antigen Pf155/RESA. A plasmid vector,pSPPM3ABPXM is constructed, in which a gene fragment encoding SEBinstead of M3 is introduced between the propeptide region and the ABPsequence of plasmid pSPPmABPXM. An oligonucleotide linker(5′AGCTTGGCTGTTCCGCCATGGCTCGAG-3′ with complementary sequence) isinserted into the HindIII site of plasmid pSZZmpISX thus creatingadditional NcoI and XhoI recognition sites downstream of the HindIIIsite in the resulting vector, pSZZmpI8XhoXM. A gene fragment encoding a198-amino-acid ABP from the serum albumin binding region ofstreptococcal protein G is generated by a PCR5′-CCGAATTCAAGCTTAGATGCTCTAGCAAAAGCCAAG-3′ and5′-CCCCTGCAGTTAGGATCCCTCGAGAGGTAAAATTTCATC-3′

[1565] respectively) with plasmid pSPGI as template sequenced in plasmidpRIT28 by solid-phase DNA sequencing and HindIII-XhoI subcloned in framedownstream of the mp18 multilinker of pSZZmp18XhoXM. yielding plasmidpSZZmpI8ABPXM. An M3-encoding gene fragment was BamHI-HindIII subclonedfrom plasmid pRIT28EM3DAStop into pSZZmp18ABPXM, yielding plasmidpSZZM3ABPXM. Plasmid pLipPS17 is constructed from pLipPSlk theintroduction of a BsmI recognition site in the beginning of the lipasesignal sequence. a Bc/I site at the end of the signal sequence and aBgIII site at the end of the propeptide-encoding region by site-directedin vitro mutagenesis. A gene fragment constituting almost the entire S.carnosus vector pLipPSI except for a fragment encoding the C terminus ofthe propeptide and the majority of the mature lipase from S. hyicus isisolated by SalI-Hind III digestion and ligated to the E. Co/i plasmidpRIT28. which had previously been cut with the same restrictionendonucleases. The resulting plasmid, designated pSDLip, contained theorigin of replication for both E. coli and S. aureus. To restore theC-terminal region of the lipase propeptide, a gene fragment encoding theC-terminal part is generated by PCR amplification with theoligonucleotides 5′-CCGAATTCTCGAGGCTCCTAAAGAAAATAC-3′ and5′-CCAAGCTTGGATCCTGCGCAGATCTTGGTGTTGGTTTTTTG-3′

[1566] as upstream and downstream primers. respectively, with plasmidpLipPS17 as template. This amplification introduced upstream EcoRI andXhoI sites and downstream FspI. BamHI. and HindIII recognition sequencesby noncomplementary sequences in the PCR primers. The gene fragmentencoding the C-terminal propeptide region was EcoRI-HindIII subcloned topRIT28 to verify a correct sequence by solid-phase DNA sequencing andthereafter XhoI-BamHI transferred to SalI-BamHI-restricted pSDLip. Theresulting plasmid. pSPP is HindIII restricted, filled in with Klenowpolymerase. and religated to yield plasmid pSPPDHind, which encodes thesignal peptide and the complete propeptide of the S. hyicus lipase withtranscription from a promoter region suitable for overproduction in S.carnosus.

EXAMPLE 38 Gene Transfer for Expression of an Mono orDigalactosylceramide by Transfection with a Cosmid Genomic LibraryPrepared from a Cell Line in which the Specific Glycosylceramide isHighly Expressed

[1567] The deliberate transfer of mono or digalactosylceramideexpression in tumor cells is achieved by transfection with a cosmid DNAlibrary prepared from Fabry's cells in which the mono ordigalactosylceramide is highly expressed. This model demonstrates ageneral method for transferring glycosyltransferase genes and otherfactors necessary for the expression of glycosphingolipid antigens. Therecipient tumor cells contain mono or digalactosylceramide and thedirect precursor, lactosylceramide. The transfected cells express monoor digalactosylceramide detected both chemically and immunologically andcontained human DNA detected by an Alti sequence probe.

[1568] Cells and antibodies: Fabry's cells or normal cells with an α-galactosidase deficiency and tumor cells including but not limited toneuroblastoma cells are used. Anti-galactosyl ceramide monoclonalantibody is prepared. Total DNA is prepared from Fabry's cells isexcised by MboI and ligated by Bam HI-treated cosmid vector PCV 108,which has the SV40 promotor fused to the neomycin phosphotransferasegene. The target DNA for cosmid cloning is purified by gelelectrophoresis between 30-40 KB size. In vitro packaging is made withan extract of lysogenic bacteria and propagated in E. coli. as describedelsewhere.

[1569] Transfection and Selection of Galactosylceramide Expression:Cosmid library DNAs are transfected into various cells using the calciumphosphate DNA precipitation technique with the addition of a glycerolshock after a 6 hour incubation. Galactosylceramide selection is started2 days later at 400 mg/ml concentration. The expression ofgalactosylceramide in the original Fabry's cell and the transfectedtumor cells was determined by cytofluorometry (FACS II), in whichFITC-conjugated anti-mono or digalactosylceramide antibody is used.Glycolipids in transfected cells are analyzed after cells were extractedin chloroform-methanol (2:1 and 1:1 v/v). The neutral glycolipidfraction is prepared by an acetylation procedure. The glycolipid profileis confirmed on HPTLC, followed by immunostaining with anti- mono ordigalactosylceramide antibody.

EXAMPLE 39 Staphylococcal Collagen Binding Adhesin Nucleic AcidsTransfected into SAg Transfected Tumor Cells, SAg Transfected DCs orAccessory Cells and S/D/t Cells

[1570] Collagen gene fragments from S. aureus strain FDA 574 areoverexpressed in E. coli using the vector pQE-30 (QIAGEN inc. Chatworth,Calif.). Recombinant proteins expressed from this vector contain anNH2-terminal tail of six histidine residues. The gene named cna encodinga S. aureus collagen adhesin is isolated from a S. aureus genomiclibrary cloned and sequenced. The cna gene encodes a 1185 amino acidpolypeptide. The deduced amino acid sequence reveals several structuralcharacteristics similar to previously described Gram-positive bacterialcell surface proteins.

[1571] Plasmids expressing cna gene fragments are produced as follows.Recombinant S. aureus collagen adhesin fragments are overexpressed in E.coli using three different prokaryotic expression systems. The aminoterminus including the entire A domain is amplified from S. sureus FDA574 chromosomal DNA using PCR together with primers CNA 20 and CNA 21.The amplified 1.6-kb cna gene fragment is cleaved with EcoRI and PstI,gel purified and ligated to the prokaryotic expression vector pKK223-3obtained from Pharmacia LKB Biotechnology to create plasmid pKK1.5.Expression vector pKK223-3 contains an IPTG-inducible tac promoteradjacent to a consnesus Shine-Dalgarno ribosomal binding site. However,this vector lacks an initiation codon; therefore, the DNA to beexpressed must contain an appropriate start codon. In order to expressan internal cna fragment, a DNA linker sequence containing an ATG startcodon is synthesized. Two partially complementary ologonucletides, JPI(5′AATTACCATGGAATTCCTGCA-3′) and JP2 (5′-TGGTACCTTAAGG-3′), are heatedto 70° C. and slowly cooled to allow annealing. Once annealed, thedouble-stranded linker is phosphorylated by the addition of ATP and T4polynucleotide kinase. The DNA linker contained EcoRI and PstIrestriction sites at the 5′- and 3′-termini, respectively. These sitesare used to insert the linker onto pKK223-3. A 2.9-kb EcoRI/PstI DNAfragment, originally isolated from lambdaGT11 clone pCOL1 1 was ligatedto vector pKK223-3 to create plasmid pKK2.9. The collagen adhesinfragment encoded by pKK2.9 contains three repeated domains (B1, B2, andB3), the carboxyl terminus and downstream sequences.

[1572] The plamid containing the collagen adhesin is transfected intoDTES by methods in Example 1 and 3 and expression of the transduced geneis monitored by Immunoblots (Example 33).

EXAMPLE 40 Transfection of Nucleic Acids Encoding SAgs in Combinationwith Nucleic Acids that Promote Apoptosis Induction or Predispose toApoptosis

[1573] SAgs expressed in apoptotic tumor cells or tumor cell/DC hybridsare ingested by DCs which present them to the immune system in morewhich evokes a potent immune response to the tumor associated antigens.The apoptotic cell is also one which is overexpresses a GalCer such asone with a natural or acquired α-galactosidase deficiency or from apatient with Fabry's Disease. The apoptotic stimulus can be produced byconcordant influenzal infection, radiation or chemotherapy. In addition,it may be inducible by an exogenous source such as TNF if the cell ispredisposed by transfection of an potent inhibitor of NF-kB such as amodified form of IκBα. Additional stimuli to apoptosis are provided bynumerous well established activators (caspase 9) or initiators (caspase8) of the caspase system or the CD95 TNFR network. Having undergoneapoptosis, the SAg transfected, GalCer overproducing cell is nowingested by DCs which are cross-primed to present the tumor antigens andthe GalCer in the context of SAg stimulation resulting in a potentantitumor response. Methods and protocols for SAg transfection are givenin Example 1 and for priming of DCs in Example 27-28 The apoptotictransfectants are used as a preventative or therapeutic antitumorvaccine by protocols in Example 15, 16, 18-23 and 29. They are alsouseful ex vivo to a population of tumor specific effector T cell or NKTcells for use in the adoptive immunotherapy of cancer (Examples 2-5, 7,15, 16, 18-23, 29).

EXAMPLE 41 Preparation and Isolation of Glycosphingolipids andVerotoxins

[1574] Galabiosylceramide, Globotrioslceramides andGlobotetraosylceramide

[1575] Globotrioslceramides (GB3) and globotetraosylceramide (Gb4) arepurified from human renal tissue. Briefly, the chloroform/methanoltissue extract is first applied on a Bio-Sil A (Bio-Rad) silica columnin chloroform. The column is extensively washed with chloroform, andneutral glycolipids are eluted with acetone/methanol,9:1 (vol/vol). Theneutral glycolipid fraction is then applied on a second Bio-Sil A columnin chloroform/methanol, 98:2 (vol/vol). Glycolipids are then resolvedwith a linear solvent gradient comprising equal weights ofchloroform/methanol 15:1 (vol/vol), to chloroform/methanol, 4:1(vol/vol). Galabiosylceramide (Gb2) or Gal(α1-4)Gal ceramide from marinesponge may be obtained, for example, from Dr T. Matsubara (Department ofChemistry, Kinki University, Kowakae. Japan).

[1576] VTs and Subunits: A simple method for purifying E. coli H30verocytotoxin is as follows. The toxin, released from the cells byexposure to polymyxin B, is subjected to differential ammonium sulfateprecipitation and sequential chromatography on hydroxylapatite,chromatofocussing, Cibachron blue, and Sephadex G-100 columns. Thepurified toxin, 39 kDa by gel filtration and having a pI of 6.72,resolves as a band which migrates at 32 kDa and another band of lessthan 14 kDa which migrates with the buffer front on reducing SDS-PAGE.The purified preparation is relatively heat-stable, and has a specificactivity of 3×10⁹ CD50 units/mg protein in Vero sells, and LD50 valuesof 0.2, 9.0, and 40 g protein/kg in rabbits, rats, and mice,respectively. Antiserum to the toxin specifically neutralizes H 30 VT,Shiga toxin, and VT activity from some clinical isolates of VT+E. colibut not that from a porcine edema disease strain.

[1577] Verocytotoxin 2 (VT2) is purified from E. coli strain E32511using, as starting material, cells harvested from a Penassay brothculture incubated for 6 h at 37° C. in the presence of mitomycin C (0.2mg /ml). A crude extract of VT2, is obtained by polymyxin B treatment ofcell pellets, is purified using differential ammonium sulphateprecipitation, and sequential column chromatography. The purified toxinis estimated to have a pI of 6.5 by chromatofocusing and a molecularweight of 42 kDa by gel filtration; it has a specific activity of1.39×10⁶ CD50 units/mg protein in Vero cells, and resolves as a majorband of Mr 35 kDa and another band of <14 kDa which migrates with thebuffer front on reducing SDS-PAGE. The purified toxin is not neutralizedby VT1 antisera, and antisera prepared to this toxin in rabbits did notneutralize VT1, but completely neutralized the activity of thehomologous toxin.

[1578] Recombinant Methods of Preparing VT's and Subunits: RecombinantVT1 is purified from pJLB28. VT2 from R82. and VT2c from E32511. Therecombinant E. coli strain pJLB28 is used as a source of VT1 B subunit.High yields of the toxins or subunits (10-15 mg/3 liters, of brothculture) are purified by a method involving polymyxin B extraction,ultrafiltration, hydroxylapatite chromatography, chromatofocusing, andCibacron Blue chromatography. VT2 is purified by virtually the samemethod from an E. coli clinical isolate, strain E32511. The cistronencoding the B subunit of E. coli Shiga-like toxin I (SLT-I) is clonedunder control of the tac promoter in the expression vector pKK223-3 andthe SLT-I B subunit is expressed constitutively in a wild -typebackground and inducibly in a lacI^(q) background. E. coli TB 1 lac prorpsL ara thi f 80d LacZ D MI5 hsdR is obtained from Bethesda ResearchLaboratories (Gaithersburg, Md.). E. coli JMIO1 D lacc pro supE thi (F′traD36 IacZ A MIS pro AB lacP) is obtained from Dr. J. D. Friesen(Department of Medical Genetics, University of Toronto, Toronto,Ontario, Canada). Plasmids pTZ18R and pKK223-3 are obtained fromPharmacia. Plasmid pJLB5 consists of a 3.0 kb KpnI fragment ofbacteriophage H 19B DNA cloned in the KpnI site of pUC18. To constructplasmid pJLB34, pJLB5 is cut at the BgIII site and digested withnuclease Bal31. The ends are filled with Klenow fragment and dNTPs. Thefragment remaining after deletion is cleaved with EcoRI, and the piececarrying the SLT-I B cistron is purified by agarose-gel electrophoresis.The fragment is recovered from the gel and cloned into pUC18 cut withEcoRI and HindII. The EcoRI-HindIII fragment is cloned in M13mp18 andits nucleotide sequence is determined. The B cistron coding sequence isrecovered from pJLB34 as a 1.1 kb PstI fragment and was then cloned inthe PstI fragment and was the cloned in the PstI site of the polylinkerof pKK223-3. Clones with the correct orientation of insertion relativeto the tac promoter are identified by restriction-endonuclease analysis.One plasmid with the orientation is selected and designated pJLB120.pJLB120 is transformed into E. coli TB1 for constitutive expression andinto E. coli JM101 for inducible expression. Bacteria are grown inL-broth or brain heart infusion broth (Difco Laboratories, Detroit,Mich.) supplemented as necessary with carbenicillin at 50 mg/ml and IPTG(Bethesda Research Laboratories) at 1 mM.

[1579] Expression of Toxins: For E. coli JM 101 (pJLB 120), an overnightculture is used to inoculate fresh L-broth supplemented withcarbenicillin (50 pg/ml) and was grown to mid-exponential phase(A600=0.3-0.6) at 37° C. with shaking at 300 rev./min. IPTG is added toa final concentration of 1 mM. and incubation is continued withaeration. For E. coli TB I (pJLB120), an overnight culture is used toinoculate fresh L-broth supplemented with carbenicillin (50 mg/ml), andthis is grown for 12-18 h at 37° C., with shaking at 300 rev./min. Inboth cases the culture is harvested and the pellet is washed once withPBS (0.15M-NaCl/10 mM sodium phosphate buffer, pH 7.4) beforeextraction.

[1580] Polymxin B extraction of Toxins: The washed pellet is resuspendedin PBS containing 0.1 mg/ml polymyxin B in one-quarter of the originalculture volume and extracted as previously described. For purification,18 h cultures of E. coli TBI (pJLB 120) are extracted with polymyxin B,and the extracts are concentrated 10-fold using a stirred-cell Amiconconcentrator with a Ym-5 membrane (Amicon Corp., Danvers, Mass., USA).

[1581] Quantification of Toxins: Periplasmic extracts of VT-producingclones, prepared by polymyxin B extraction, are diluted as required andfiltered onto nitrocellulose paper in a slot-blot apparatus (Bio-RadLaboratories). VT is detected by using MAb 1 3C4 according to theWestern-blot procedure described above. Blots are scanned with aMolecular Dynamics model 300A computing densitometer. VT is quantifiedby comparison with a standard curve generated with purified B subunitprotein.

[1582] Purification of Toxins: The concentrated polymyxin B extract aredialysed overnight against 50 mM-Tris/HCl buffer, pH 7.4, and thenapplied to a DEAE-Sephacel column (1 cm×20 cm) equilibrated with 1mM-Tris/HCL buffer, pH 7.4. Bound material is eluted by using a lineargradient of 0-1M-NaCl in 50 mM-Tris/HCl buffer, pH 7.4, and 5 mlfractions are collected. Fractions containing VT are identified, pooledand concentrated with Centriprep-3 concentrators (Amicon Corp.). Thispool is dialyzed overnight against 25 mM-imidazole/HCl buffer, pH 7.4,and is applied to a column (1.5 cmx 2° Cm) of Polybuffer exchangcr 94(Pharmacia) equilibrated with the same buffer. Elution is carried outwith a degassed solution of Polybuffer 74 (Pharmacia) diluted 1:8 withdistilled water and adjusted to pH 4.0 with HCl (11 column volumes).Fractions (5 ml) are collected, and the B subunit positive fractions arepooled and concentrated with Centriprep-3 (Amicon). Ampholytes areremoved by Sephadex G-50 gel-filtration.

[1583] HPLC Purification of Toxins Approximately 1 mg (in 1 ml) ofpurified toxin or subunit is injected into a TSK-G2000SW HPLC gelfiltration column previously equilibrated with 50 mM Tris-bufferedsaline (TBS), pH 7.4, flow rate of 1.0 ml/mm. Peaks, measured byabsorbance at 1=280 nm, arecollected.

[1584] Toxin Subunit Separation: 1 mg of toxin subunit is concentratedto 30-50 ml using a Centricon 30 concentrator (Amicon). 1 ml of asubunit dissociating solution (6 M urea, 0.1 M NaCl, 0.1 M propionicacid, pH 4, is added dropwise, and the toxin is incubated withoutstirring at 4° C. for 1 h. The solution is then separated by HPLC gelfiltration (as above) after previous column equilibration with thedissociating solution. Peaks, measured by absorbance at 1=280 nm, arecollected.

EXAMPLE 42 Gangliosides Shed from Tumor Cells: Isolation from Tumor CellSupernatants

[1585] Collection of Tumor Cell Supernatant

[1586] Tumor cells are cultured in 25 ml of no serum-low protein medium(NSLP) in an 8° Cm² flask for 1-5 days. Cells are harvested bycentrifugation at 400 g for 10 mm, and the supernatant is concentrated10-fold at 4° C. in an Amicon stirred cell with a 10-kDa cutoffultrafilter. Concentrated supernatant and NSLP concentrated under thesame conditions are stored at −20° C., and passed through a 0.1-umsterile membrane filter.

[1587] Metabolic Labeling of Gangliosides in Tumor Cell Supernatant

[1588] Tumor cells (10⁵/ml) are cultured in 10 ml of NSLP for 2 days.After three washes with fresh medium, cells are transferred into 10 mlof NSLP containing 1 mCi/ml D-[1-¹⁴C]GlcNH2-HCl (50 mCi/mmol (ICNBiomedicals, St. Laurent, Quebec, Canada) and 1 mCi/ml of D-[1-1⁴C]Gal(56 mCi/mmol; Amersham) to label gangliosides. After 24 hr, cells arewashed with medium three times to remove unincorporated sugars, thencultured for an additional 24-48 hr in fresh medium, before harvestingby centrifugation at 400 g. Radioactivity in the tumor cell supernatantand cells is quantitated by liquid scintillation counting. Thesupernatant is clarified by centrifugation at 15,000g for 10 mm, thenconcentrated 10-fold using a Speedvac concentrator, before beinganalyzed by gel filtration chromatography.

[1589] Gel Filtration Chromatography of ¹⁴C-Labeled Tumor CellSupernatant on Sepharose 2B-300

[1590] Concentrated ¹⁴C-labeled tumor cell supernatant ischromatographed on a Sepharose 2B-300 column (5 ml bed volume; SigmaChemical Go, St Louis, Mo.), equilibrated with Tris-buffered saline(TBS; 50 mM Tris-HCl in 0.15 M NaCl, pH 7.4). The column is eluted at aflow rate of 0.2 ml/min at 22° C., and 200 ml fractions are collectedand counted for ¹⁴C. Dipalmitoylphosphatidylcholine liposomes and sodiumazide are used as standards to calibrate the void and included volume ofthe column, respectively.

[1591] Gel Filtration FPLC of ‘P-Labeled Tumor Cell Supernatant onSuperose

[1592] FPLC is carried out on a Superose 6 column (1×30 cm; Pharmacia,Dorval, Quebec, Canada) linked to a Gilson HPLC system and a Gilson iliBultraviolet flow detector. The column is calibrated with a series ofstandard proteins of known molecular mass, ranging from β-galactosidase(465 kDa) to β-lactoglobulin (36.8 kDa) (Pharmacia, High MolecularWeight Gel Filtration Calibration kit). The void volume and includedvolume are determined using Blue Dextran (2000 kDa) and sodium azide,respectively. ³H-Labeled bovine brain gangliosides and [¹⁴C]Galdissolved in NSLP or TBS are also used as standards. Concentrated YAC-1supernatant is eluted through the column at 22° C. with TBS at a flowrate of 0.5 ml/min. Fractions (0.5 ml) are collected and counted for¹⁴C.

EXAMPLE 43 Assessment of SAg and VT Binding to Glycosphingolipids by TLCOverlay

[1593] Glycolipids (dissolved in chloroform/methanol (2:1 v/v), areapplied to a TLC plate and separated in choroform/methanol/water(65:25:4, v/v). Toxin binding is determined using known methods.Briefly, after separation of the glycolipids, the plate is air dried,incubated overnight at 37° C. in a solution of 1% (m/v) gelatin in 50 mMTris/HCL, 150 mM NaCI, pH 7.4 (buffer A). The plate is washed in bufferA and incubated successively with VT 1 (0.07 mg/ml in buffer A) followedby monoclonal antibody PHI (1.5 mg/ml in buffer A), and finally withgoat antimouse IgG horseradish peroxidase conjugate (diluted 1:2000 inbuffer A). Toxin binding is visualized using 4-chloro-1-naphthol. Anequivalent plate is run and treated with 3% (m/v) orcinol spray in 3 MH₂SO₄ to visualize carbohydrate and ensure equal concentrations.

[1594] Alternate Microtitre Plate Binding Assay

[1595] Quantification of toxin binding to various glycoconjugates isperformed using published methods. A methanolic solution [100 plcontaining glycolipid (300 nmol). phosphatidylcholine (0.5 mg) andcholesterol (0.25 mg)] is added to microplate wells and the methanol isallowed to evaporate overnight at room temperature. The wells areblocked with 2% (m/v) BSA in buffer A (200 ml/well) for 2 h at roomtemperature and subsequently washed once with buffer A containing 0.1%BSA (BSA/buffer A). 100 ml aliquots of dilutions of [¹²⁵I]-VT-1 inBSA/buffer A are added to the wells and incubated for 2 h at roomtemperature. The wells are washed five times with BSA/buffer A, excisedand the radioactivity is measured in a g counter. Scatchard analysis wasperformed using the LIGAND program.

EXAMPLE 44 Methods of Induction and Assessment of Apoptosis & Inhibitionof Protein Synthesis

[1596] Tumor cells (5×10⁵cells/ml) are cultivated at 37° C. in 96-wellround-bottomed microtiter plates (Becton Dickinson) in 200 mlleucine-depleted RPMI (Eurobio, France) containing 1 mCi of [³ H]leucine. with or without 10 ng/ml VT. After 18 hrs. cells are harvestedon class fiber filters, and radioactivity incorporated in proteinsmeasured in a scintillation counter.

[1597] Ultrastructural Analysis of VT-Treated Astrocytoma Cells

[1598] Cells are cultivated on a transferable 9 mm cylcopore membrane(0.45 mm pore size. Falcon) to form a confluent monolayer and areincubated at 37° C. with VTI (10 ng/ml). Cells are fixed at roomtemperature by addition of 1.6% glutaraldehyde to the wells and thenincubated in 0.066 M Sorensen buffer (pH 7.4) containing 1.5%glutaraldehyde for 1h at 4° C. After 2 h of washing with 0.1 phosphatebuffer, cells are post-fixed in 2% osmium tetroxide in the same buffer.After dehydration in graded ethanols and propylene oxide, Eponembedding, thin sectioning and uranyl-lead counterstaining on grids areperformed. Thin sections are examined in a Philips EM 400 electronmicroscope and ultrastructural features of apoptosis are analyzed

[1599] Flow cytometry: Apoptosis of astrocytoma cells, incubated with 10ng/ml of VT1 for 24-36 hrs in-the presence of 10% bovine fetal serum isanalyzed on an Epics Profile Analyzer (Coulter Electronics. Pathology.University of Toronto) according to known procedures. After treatment,cells are trypsinized and the 200×g centrifuged cell pellet is suspendedin.1 ml of hypotonic fluorochrome solution of 50 mg/ml propidium iodide(Sigma) and stained for 30 min at 4° C. To remove RNA prior to staining.cells are treated with 100 ml of 200 mg/ml solution of DNase-free RNaseA at 37° C. for 30 min. Cell cycle distribution is determined usingmanual gating. Flow cytrometric quantitation of apoptotic cells withinthe propidium iodide-stained population is performed as described.Debris and dead cells are excluded on the basis of their forward andside light-scattering properties. Astrocytoma cells grown simultaneouslyin the absence of VT1 serve as controls.

[1600] DNA Fragmentation Assays Cells: Tumor cells are incubated in RPMI1640 medium alone or in the presence of intact VT or VT-B. After 18-hculture, cells are counted and viability assessed by trypan blueexclusion. Cells are then centrifuged and washed twice with salinebuffer. The pellets are lysed by incubation for 1 h at 50° C. in 10 mMEDTA, 200 mM NaCl, 0.1 mg/ml proteinase K, 0.5% (w/v) SDS, and 50 MmTris-HCL, pH 8. The DNA is extracted with phenol,chloroform:isoamylalcohol (24:1), and then ethanol precipitated.Unfragmented DNA is discarded, and 0.1 volume of 3 M sodium acetate, pH7.2, is added to the supenatant which is left at −80° C. overnight. Theprecipitate containing fragmented DNA is centrifuged (1300 g, 30 mm) anddried under vacuum. DNA derived from 5×10⁶ cells is then resuspended in20 ml RNAse buffer containing 0.5 mg/ml DNAse-free RNAse (Sigma), 15 mMNaCl, and 10 mM Tris-HCL, pH 7.5. and incubated at 50° C. for 1 h;Electrophoresis is carried out at 70V in 2% agarose gel containing 0.1mg/ml ethidium bromide in a buffer containing 2 mM EDTA, 80 mMTris-phosphate. pH 8. After electrophoresis. gels are examined under UV.Phage DNA from bacteriophage 1 and f digested by HindIII and HaeII,respectively, provide molecular weight standards.

[1601] Nuclear staining with propidium iodide: SF-539 cells grown on thecover slips overnight are incubated at 37° C. with VT-12B subunit (50mg/ml) for 1.5 hrs or 10 hrs and fixed (with 1% paraformaldehyde for 3minutes). permeabilized with 0.1% Triton X in 100 mM PBS for 5 min, andstained with 5 mg/ml propidium iodide (Sigma). After extensive wash with50 mM PBS, the fixed cells are mounted with DABCO(1,4-diazabicyclo-octane (Sigma), and nuclear staining is observed underincident UV illumination.

[1602] Proliferation assay: Approximately 1-5×10⁴ cells are added to24-well plates and incubated in a-MEM in 5% CO2 at 37° C. After 24 hr,the growth medium is replaced with medium containing variousconcentrations of the holotoxin VT1 (0.0.1.5.50, 100 ng/ml). The treatedastrocytoma cell lines and endothelial cells are trypsinized and countedat intervals throughout the growth curve. Cell viability is assessed bytrypan blue dye exclusion. Cell counts are plotted against time for thevarious concentrations of VT1 and B subunit. For each time pointanalyzed, the wells are set-up in triplicate.

[1603] For selected cell lines, the B subunit of VT1, VT2, and VT2c isadded alone to the astrocytoma cells at same concentrations listedabove. A single dose of VT1. VT2. and VT2c is added to confluentastrocytoma cells in microplate wells. Cell survival at 72 hr ismonitored by staining with 0.1 % crystal violet, and measuring theoptical density at 590 nm using a Dynatek microtiter plate reader.

EXAMPLE 45 Multidrug Resistant Cells: Culture and Preparation

[1604] MCF-7-wt and MCF-7-AdR (adriamycin-resistant) cells are obtainedfrom Drs. K. H. Cowan and M. B. Goldsmith, National Cancer Institute.Cells are maintained in RPMI 1640 medium containing 10% FBS (v/v), 50units/ml penicillin, 50 mg/ml streptomycin, and 584 mg/literL-glutamine. KB-3-1 human oral epidermoid carcinoma cells (parent,drug-sensitive) and KB-V-1 cells (highly MDR) and subclones are obtainedfrom the National Cancer Institute). Cells are grown in high glucose(4.5 g/liter) Dulbecco's modified Eagle's medium containing 10% FBS andother components described above. The KB-V-1 cell line is maintainedwith vinblastine (1.0 mg/ml) in the medium. NIH:OVCAR-3 cells (humanovarian adenocarcinoma, drug-resistant) are obtained from the AmericanType Culture Collection and grown in RPMI 1640 medium containing insulin(10 mg/ml), 10% PBS, and other components listed above. All cells arecultured in a humidified, 6.5% CO2 atmosphere, tissue culture incubator.Cells are subcultured once a week using 0.05% trypsin and 0.53 mM EDTAsolution.

[1605] Lipid Mass Analysis: Cell lipids are analyzed by TLC separationand charring of the chromatogram. Briefly, total cellular lipids areextracted and equal aliquots (by weight) from each sample are spotted onTLC plates. Plates are developed in the desired solvent system (seebelow), air-dried for 1 h, and sprayed using a 35% solution of sulfuricacid in water (v/v). The lipids are charred by heating in an oven at180° C. for 30 mm, and resulting black bands are visualized.

[1606] Cell Radiolabeling and Analysis of Sphingolipids: MCF-7 cellsgrown in medium containing 10% FBS, are switched to serum-free mediumcontaining 0.1% fatty acid-free BSA. Cell lipids are radiolabeled byincubating cells with [³H]serine (2.0 mCi/ml), [³H]palmitic acid (1.0mCi/ml, or [³H]galactose 1.0 mCi/ml) for the indicated times. In someinstances, cells are radiolabeled in medium containing 5% PBS. Cells arethen rinsed twice with PBS, and 2 ml of ice-cold methanol containing 2%acetic acid is added. The cells are scraped free, transferred to glasstest tubes (13×100 mm), and lipids are extracted by the addition ofchloroform (2 ml) followed by water (2 ml). The resulting organic lowerphase is evaporated under a stream of nitrogen. Lipids are resuspendedin 100 ml of chloroform methanol (1:1, v/v) and aliquots are applied toTLC plates. When using [³H]galactose, radiolabeled cells are washedtwice with PBS, transferred to glass tubes with methanol (2 ml, andglucosylceramides and gangliosides (2.5 mg of each) are added to aidrecovery. Lipids are extracted by the addition of water (2 ml; and 2 mlof chloroform (three times consecutively). The pooled organic lowerphase is treated as above. Lipid analysis is carried out by various TLCseparations using solvent system I, chloroform/methanol/ammoniumhydroxide (65:25:5, v/v); solvent system II,chloroform/methanol/ammonium hydroxide (40:10:1, v/v), solvent systemIII, chloroform/methanol/water (60:40:8, v/v), or solvent system IV,chloroform/methanol/acetic acid/water (50:30:7:4, v/v). Fordetermination of ceramides. an aliquot of the chloroform-soluble lipidsis base-hydrolyzed in 0.1 N KOH in methanol for 1 h at 37° C.; lipidsare re-extracted and separated using solvent system V hexane/diethylether/formic acid (60:40:1, v/v). Galactosyl- and glucosyl-ceramides areseparated using solvent system VI, chloroform/methanol/water (60:25:4,v/v). This separation is performed on TLC plates that are pre-run in2.5% borax in methanol/water (1: 1) and heated at 110° C. prior to use.

[1607] Radiochromatograms are sprayed with EN³HANCE and exposed for 3-7days for autoradiography. TLC areas, aligned with hands on theautoradiographs or with iodine-stained commercial lipid standards arescraped from the plate. Water (0.5 ml) is added to the plate scrapings,followed by 4.5 ml of EcoLume counting fluid, and the samples arequantitated by liquid scintillation spectrometry.

[1608] Purification of Glycosylceramides: The compounds, extracted withtotal lipids from MCF-7-AdrR cells, are resolved from other lipids onpreparative TLC using silica gel H plates developed in solvent systemII. The appropriate region of the TLC plate is then scraped into testtubes, and lipids are extracted with chloroform/methanol/aceticacid/water (50:25:1:2, v/v). The samples are centrifuged, and thesolvent transferred to new glass tubes and evaporated to dryness undernitrogen.

[1609] Fast-Atom Bombardment/Mass Spectrometry of TLC-isolated Lipid-:FAB/MS spectra are acquired using a VG 70 SEQ tandem hybrid instrumentof EBqQ geometry (VG analytical, Altrincham, UK.). The instrument isequipped with a standard unheated VG FAB ion source and a standardsaddle-field gun (Ion Tech Ltd., Middlesex, UK) that produces a beam ofxenon atoms at 8 kV and 1 mA. The mass spectrometer is adjusted to aresolving power of 1000, and spectra are obtained at 8 kV using a scanspeed of 10 s/decade. 2-Hydroxyethyl disulfide is used as matrix in thepositive FAB/MS, and triethanolamine is used as a matrix in the negativeFAB/MS. Negative FAB and positive FAB give different values for the samecompounds, due to charge (proton content) differences.

EXAMPLE 46 Incubation of Tumor Cells with Hydroxy Fatty Acids forSelective Synthesis of Galactosphingolipids and Lipid Analysis

[1610] Tumor cells on filters are incubated for 1 hr at 37° C. in thepresence of labeled and unlabelled [³H]Cer(C6[D-20H]). After theincubation, lipids are extracted from the cells and the combinedincubation media and analyzed Lipids are extracted from cells and mediaby a two-phase extraction. The upper phase contains 20 mM acetic acidand (for radiolabeled lipids) 120 mM KCl. After a chloroform wash. whichis added to the lower phase, lipids remaining in the upper phase GalCerare collected on SepPak C18 cartridges (Waters, Milford, Mass.) fromwhich lipids are eluted with chloroform/methanol/water 1:22:0.1) andmethanol. The organic (lower) phase is dried under N₂, and the lipidsare applied to TLC plates that were dipped in 2.5% boric acid inmethanol, dried, and activated by heating at 110° C. for 30 mm. They aredeveloped in two dimensions:

[1611] I. chloroform/methanol/25%NH4OH/water (65:35:4:4. v/v); and

[1612] II. chloroforrm/acetone/methanol, acetic acid/water(50:20:10:10:5. v/v).

[1613] Fluorescent spots are detected under UV scraped from the TLCplates and the fluorescent lipid analogs are extracted from the silicain 2 ml chloroform/methanol/20 mM acetic acid (1:2:2:1 v/v) for 30 mm.After pelleting the silica for 10 min at 1,500 rpm fluorescence in thesupernatants is quantified in a fluorimeter (Kontron. Zorich,Switzerland). Radiolabeled spots are detected by fluorography afterdipping the TLC plates in 0.4% PPO in 2-methylnaphthalene with 10%xylene. Preflashed film (Kodak X-Omat S) is exposed to the TLC platesfor 3 d at −80° C. The radioactive spots are scraped from the plates,and the radioactivity is quantified by liquid scintillation counting in0.3 ml Solulyte (J. T. Baker ChemicaL. Deventer, The Netherlands) and 3ml of Ultinsa Gold (Packard Instruments. Downers Grove. Ill.).

EXAMPLE 47 Conjugation of Proteins to Lipoproteins

[1614] The preferred method for coupling superantigens to lipoproteinsis to use 10 mM solution of sodium periodate for oxidation of thecarbohydrate in the lipoprotein. This will also cleave c-c bonds in thesugars with adjacent hydroxyls and oxidize them to reactive aldehydes.Superantigens form Schiff base linkages with the aldehyde modified sugargroups under alkaline conditions. the aldehyde modified sugar is thencoupled to the amine containing superantigen peptide or polypeptide. Theoxidation is followed by reductive amination using sodiumcyanoborohydride to reduce the labile Schiff base between the aldehydeon the carbohydrate and the amine on the superantigen to form stablesecondary amine covalent linkages.

[1615] An alternative procedure is to periodate oxidize the lipoproteinas above to create reactive aldehyde groups. Heterobifunctionalcross-linking agent such as 4-(4-N-Maleimidophenyl)butryric acidhydrazide (MPBH) 4-(4-N-Maleimidophenyl)buryric acid hydrazide (MPBH),and 4-(N-Maleimidomethyl)cyclohexane-1-carboxyl-hydrazide (M2C2H) whichcontain a carbonyl-reactive hydrazide group on one end and asulfhydryl-reactive maleimide on the other are preferred. The hydrazidereacts specifically with aldehyde functional groups to create ahydrazone linkage a type of Schiff base. To stabilize the bond betweenthe hydrazide and aldehyde, the hydrazone is reacted with sodiumcyanoborohydride to reduce the double bond and form a secure covalentlinkage. The cross-bridge between the two functional ends provides along, 17.9-A spacer. These agents couple to periodate-oxidized aldehydeson the lipoportein carbohydrate via the hydrazine and to sulfhydrylgroups on the superantigen via sulthydryl reactive maleimide group.Superantigens without reactive sulfhydryl groups are first thiolatedwith SATA or Trout's reagent before addition to the reactive maleide. Asulthydryl-containing protein or molecule is is bound via the maleimideend of MPBH and the derivative purified by gel filtration to removeexcess reactants, and then mixed with a lipoprotein (that had beenpreviously oxidized to provide aldehyde residues) to effect the finalconjugation.

[1616] The opposite approach e.g., modification of the glycoproteinfirst, purification, and subsequent mixing with a sulfhydryl-containingmolecule is also acceptable. With this second option, however, thepurification step should be done quickly to prevent extensive hydrolysisof the maleimide group. (See Hermanson GT Bioconjugate TechniquesAcademic Press, San Diego Calif., 1996).

[1617] Protocol for periodate oxidation

[1618] 1. Periodate-oxidize a liposome suspension containing glycolipidcomponents according to Section 2. Adjust the concentration of totallipid to about 5 mg/ml.

[1619] 2. Dissolve the protein to be coupled in 20 mM sodium borate,0.15 M NaCl, pH 8.4, at a concentration of at least 10 mg/ml.

[1620] 3. Add 0.5 ml of protein solution to each milliliter oflipoprotein suspension with stirring.

[1621] 4. Incubate for 2 h at room temperature to form Schiff baseinteractions between the aldehydes on the lipoprotein and the amines onthe protein molecules.

[1622] 5. In a fume hood, dissolve 125 mg of sodium cyanoborohydride inI ml water (makes a 2 M solution). This solution may be allowed to sitfor 30 mm to eliminate most of the hydrogen-bubble evolution that couldaffect the lipoprotein suspension.

[1623] 6. Add 10 μl of the cyanoborohydride solution to each milliliterof the lipoprotein reaction.

[1624] 7. React overnight at 4° C.

[1625] 8. Remove unconjugated protein and excess cyanoborohydride by gelfiltration using a column of Sephadex G-50 or G-75.

EXAMPLE 48 Isolation of Lipoproteins

[1626] Human LDL is isolated by sequential ultracentrifugation (d1.019-1.063 g/ml) from freshly drawn, citrated normolipidemic humanplasma to which EDTA 0.1 mmol/liter is added. Freshly obtained plasma issubjected to differential ultracentrifugation to isolate the desiredlipoprotein fractions Typically, the following density fractions wereisolated: 1) d<1.02. to remove VLDL and IDL; 2) d=1.02-1.05, to obtainLDL; 3) d=1.05-1.08, to obtain Lp(a); and 4) d=1.08-1.21, to obtainLp(a) and HDL. The Lp(a)-containing density fractions were subjected togel filtration chromatography on a Bio-Gel A-15 m column (2.5×90 cm).This column was eluted with 1.0 M NaCl, 10 mM Tris, 10 mM NaN3, 1 mMEDTA, pH 7.4, and was continuously monitored at 280 nm. The LDL- andHDL-containing density fractions are also subjected to gel filtrationchromatography to remove any contaminating species and for uniformity ofsample preparation. They are further dialyzed against 0.01 M sodiumphosphate pH 7.4, containing 0.15 M sodium chloride and 0.01% EDTA,sterilized on 0.2-um Millipore membrane, and stored at 4° C. undernitrogen (up to 3 weeks).

[1627] Lipoprotein (a) (Lp(a)): Lp(a) is prepared from fresh humanplasma by flotation centrifugation followed by affinity chromatographyon lysine-Sepharose and CsCI density gradient centrifugation asdescribed. Lipoprotein preparations are dialyzed against 0.15 M sodiumchloride containing 0.01% EDTA at 0.01% sodium azide, filter sterilized(0.45 pm) and stored at 4° C. in vials filled to allow no air space. Nocontamination of the preparations by plasminogen is detected by eitherCoomassie Blue staining of sodium dodecyl sulfate (SDS) gels or bytreatment with streptokinase and measuring plasmin activity with achromogenic substrate. S2251. The sensitivities of these assays excludedplasminogen contamination of >1% and >0.4% respectively. Lp (a)-freeLDL, HDL and acetylated LDL are prepared as previously described. TheLDL contained no apoA-1 and the HDL contained no detectable apoB-100.The apoprotein composition is verified by SDS polyacrylamide gelelecrophoresis.

[1628] Lysine-Sepharose Chromatography: Lipoprotein (a) has an affinityfor lysine-Sepharose by virtue of lysine binding kringle 4 domain(s)located on apo(a). The most important domain appears to be kringle 437,which has the greatest homology to kringle 4 of plasminogen, althoughthere may be other kringles with lesser affinity for lysine which alsocontribute to the interaction of Lp(a) with lysine-Sepharose.Plasminogen and Lp(a) have similar affinities for lysineSepharose;however, Lp(a) species with different apo(a) isoforms may haveaffinities that are significantly greater or weaker than that ofplasminogen. The buffer of choice in the isolation of plasminogen fromplasma by lysine-Sepharose affinity chromatography has been 0.1 Mphosphate buffer, pH 7.4. When the same buffer system is used in thechromatography of Lp(a), not all the lipoprotein is found to bind to thelysine-Sepharose i.e., approximately 80% of Lp(a) contained in theplasma had the capacity to interact with lysine-Sepharose. Thepercentage of Lp(a) binding to lysine-Sepharose is increased by loweringthe ionic strength of the buffer medium. Lipoprotein (a) species withlarge apo(a) isoforms tend to self-associate in the cold therefore it isbest to perform the chromatographic isolation at room temperature.

[1629] Preparation of Lysine-Sepharose 4B: Packed Sepharose 4B (250 ml)is washed with 8 liters of water on a coarse sintered glass funnel andactivated with 25 g CNBr dissolved in 50 ml acetonitrile. The reactionis carried out in a well-ventilated hood, on ice, and the pH ismaintained with 6 N NaOH at pH 11. After approximately 15 to 30 mm, theactivated Sepharose 4B is washed with 8 liters of 0.1 M NaHCO₃ pH 8.1.The agarose is then packed by filtration, diluted with 250 ml of 0.1 MNaHCO₃ pH 8.1, containing 50 g lysine, and stirred gently overnight at4°. The freshly conjugated lysine-Sepharose is then washed with 6 to 10liters of 1 mM HCl followed by 8 liters of 0.1 M NaHCO3, pH 8.1, and analiquot is saved for determination of the concentration of immobilizedlysine residues using the method of Wilkie and Landry. The concentrationof coupled lysine varies from 15 to 25 umol per milliliter packed gel.

[1630] Chromatography: Bio-Rad (Richmond, Calif.) Econo-Pac columns(1×12 cm) are packed with 5 ml lysine-Sepharose which is preequilibratedwith column buffer (e.g., 0.1 M phosphate, 0.01% NaN₂, pH 7.4). A porouspolymer filter is placed on top of the lysine-Sepharose gel bed toprevent the column from running dry. Plasma samples smaller than 3 mlare applied to the column and allowed to run through by gravity at roomtemperature. Larger volumes (up to 50 ml) should be applied with a pumpor by gravity, but at flow rates that should not exceed 20 ml/cm²/hr.The samples are washed into the column with four 0.5-ml aliquots ofcolumn buffer to be followed with four 0.5 ml aliquots, before Lp(a) iseluted withe 0.2<EACA in 10 mM phosphate, pH 7.4. One milliliteraliquots are applied at a time, and 1 ml fractions are collected inseparate tubes. Liprotein(a)and plasminogen-containing fractions (tubes4 through 10) are located by their absorbance at 280 nm. The volume ofapplied plasma depends on the Lp(a) content and on the sensitivity ofthe absorbance monitor that is part of the density gradientfractionating system.

[1631] Density Gradient Centrifugation of Lp(a): Place 5 ml of 20% (w/w)NaBr into a SW-40 ultracentrifuge tube (ultraclear). Carefully layer theeluate from the lysine-Sepharose column (up to 8 ml) on top of the NaBrsolution and, if necessary, top off the tube with 0.2 M EACA, 10 mMphosphate, pH 7.4. Place the tubes in the bucket of the swinging-bucketrotor and centrifuge 64 hr at 39,000 rpm and 20°. After centrifugationis completed, the tubes are carefully removed from the buckets andplaced in the density gradient fractionating system. The tubes arepierced at the bottom, and the gradient is pushed out the top at a flowrate of 1 ml/min with a dense fluorocarbon oil, Fluorinert FC-40 (ISCO),that has a density of 1.85 g/ml. The chart speed is 1 cm/min, and thefraction collector is set to 0.5 ml/tube. The gradient is monitored at280 nm, and the sensitivity of the chart recorder is adjusted accordingto the Lp(a) content of the eluate. Densities of the various fractionsare measured with a density meter by established techniques.

[1632] Isolation of Apolipoproteins B-48 and B-100: The followingdensity gradient ultracentrifugation procedure for isolatingsubfractions of triglyceride-rich lipoproteins is suitable for SDS-PAGEon both slab and rod gels. Plasma is recovered by low speedcentrifugation (1750 g, 20 min, 10). To minimize proteolytic degradationof apo B, 1.0 μl/ml plasma phenylmethylsulfonyl fluoride (PMSF, Sigma,St. Louis, Mo.), 10 mM dissolved in 2-propanol, and 5 μl/ml plasmaaprotinin (Trasylol, Bayer, Leverkusen, (Germany), 1400 μg/liter, areadded. Subsequently 140.4 mg solid NaCl is added per 1.0 ml plasma toincrease the density to 1.10 kg/liter. Normally, atotal volume of 4.0 mlof the d 1.10 kg/liter plasma is put in the bottom of a 13.4-mlpolyallomer ultracentrifuge tube (Ultra-Clear, Beckman Instruments, PaloAlto, Calif.). Alternatively, 3.0 ml plasma can be mixed with 1.5 ml)1.42 kg/liter NaUr, from which 4.0 ml is transferred to theultracentrifuge tube. For the rod gel method, two such tubes arerequired to obtain enough material from each sample. For the slab gelmethod, 1.0 ml plasma is sufficient. In the latter case, a 1.0 mlportion of 1.10 kg/liter plasma can be mixed with 3.0 ml of 1.10kg/liter NaCl in the tube. A density gradient consisting of 3.0 ml eachof 1.065, 1.020, and 1.006 kg/liter NaCl solutions is then sequentiallylayered on top of the plasma.

[1633] Ultracentrifugation is performed in a SW40 Ti swinging bucketrotor (Beckman) at 40,000 rpm and 15° (Beckman L8-55 ultracentrifuge).Consecutive runs calculated to float Svedberg flotation rate (Sf)>400(32 min), SI 60-400 (3 hr 28 mm), and Sf20-60 (14-16 After eachcentrifugation, the top 0.5 ml of the gradient containing the respectivelipoprotein subclasses is aspirated, and 0.5 ml of density 1.006kg/liter salt solution is used to refill the tube before the next run.The Sf1 12-20 fraction is recovered after the last ultracentrifugal runby slicing the tube 29 mm from the top after the Sf 20-60 lipoproteinshave been aspirated. All salt solutions should be adjusted to pH 7.4 andcontain 0.02 % (w/v) NaN3 and 0.01% Na2EDTA. This method yieldslipoprotein preparations almost completely devoid of plasma albumin.

EXAMPLE 49 Preparation & Isolation of Oxidized LDL (oxyLDL

[1634] Oxidized LDL (oxyLDL): Native LDL (200 μg protein/ml) is oxidizedby exposure to 5 uM CuSO4 for 24 h at 25° C. and the degree of oxidationis assessed by the increase of mobility on 1% agarose gel (1.3-1.5versus native LDL) and the formation of thiobarbituric acid-reactivesubstances (3.41±0.8 mmol/L). Oxidation is terminated by refrigeration.Different preparations of oxyLDL display similar electrophoreticmobilities. For comparison, commercially available preparations ofnative and copper-oxidized LDLs (Sigma Chemical Co., St. Louis, Mo. andBiomedical Technologies, Inc. Stoughton, Mass., respectively) are used.The level of LDL oxidation is evaluated by monitoring the formation oflipid hydroperoxides, using the FOX-2 procedure and thiobarbituricacid-reactive substances (TBARS)). The relative electrophoretic mobilityis evaluated on Hydragel (Sebia, Paris, France) and the level oftrinitrobenzenesulfonic acid-reactive amino groups was determined

[1635] The formation of thiobarbituric acid-reactive substances is 17.8nanomoles of malondialdehyde/mg protein using an oxyLDL preparation withrelative electrophoretic mobility of 1.4.

[1636] Methods for Measurement of Low-Density Lipoprotein Oxidation

[1637] Oxidation of LDL in vitro is accompanied by characteristicchanges of chemical, physicochemical, and biological properties, and avariety of methods may therefore be used for determining the extentand/or rate of oxidation of LDL. They include measurement of theincrease of thiobarbituric acid-reactive substances (TBARS), total lipidhydroperoxides defined lipid hydroperoxides, hydroxy and hydroperoxyfatty acids, conjugated dienes, oxysterols, lysophosphatides, aldehydesand fluorescent chromophores as well as measurements of thedisappearance of endogenous antioxidants and polyunsaturated fattyacids, and oxygen uptake. The apolipoprotein B (apoB) becomesprogressively altered during oxidation; its loss of reactive aminogroups and fragmentation to smaller peptides is determined and used asan index of oxidative modification. The net increase of the negativesurface charge of the whole LDL particle is analyzed as relativeelectrophoretic mobility (REM) by agarose gel electrophoresis. Thebiological assays used most frequently for assessment of the extent ofoxidative modification are the rate of uptake of LDL by culturedmacrophages and its cytotoxicity toward cultured cells. Immunologicalassays such as enzyme-linked immunosorbent assay (ELISA) andradioimmunoassay (RIA) employing polyclonal or monoclonal antibodiesrecognizing certain modifications in apoB characteristic for oxidativemodification are employed. The epitopes produced by covalent binding ofmalonaldehyde or 4-hydroxynonenal are of particular interest. Nuclearmagnetic resonance (NMR), electron spin resonance (ESR), circulardichrorism (CD), and fluorescence polarization have also been applied tostudy certain aspects of LDL oxidation. Simple methods, such as themeasure of TBARS, conjugated dienes, or fluorescence are preferred. Mostcharacterize oxidized LDL by at least two independent measurements, forexample, TBARS or REM and macrophage uptake, antioxidants and conjugateddienes.

[1638] From kinetic experiments one can conclude that both cell-mediatedoxidation of LDL and oxidation in the absence of cells catalyzed by Cu²⁺ions proceed in three consecutive time phases: lag phase, propagationphase, and decomposition phase. I)during the lag phase the LDL becomesdepleted of antioxidants, and during this period only minimal lipidperoxidation occurs in LDL, as shown by measuring polyunsaturated fattyacids (PUFAs), TBARS, lipid hydroperoxides, fluorescence, and conjugateddienes. When LDL is depleted of its antioxidants, the rate of lipidperoxidation rapidly accelerates and a lipid peroxide maximum is reachedafter about 70-80% of the LDL, PUFAs are oxidized. Thereafter, theperoxide content of LDL, starts to decrease again because ofdecomposition reactions. During the lag and propagation phases the timeprofile for TBARS, fluorescence at 430 nm, lipid peroxides, dienes, andREM are very similar and only after the peroxide maximum do thedifferent indices separate and follow different kinetics. This alsoindicates that all the methods will give equivalent results for thesusceptibility of LDL to oxidation as measured by the duration of thelag time.

[1639] Preparation of Low-Density Lipoproteins for Oxidation Isolationof Low Density Lipoproteins

[1640] After overnight fasting blood samples are withdrawn byvenipuncture and collected by free flow of blood into plastic tubescontaining the appropriate volume of an aqueous solution of 10% EDTA(w/v) (disodium salt, pH 7.4) to obtain a final blood concentration of0. 1% EDTA (wlv). EDTA serves as anticoagulant and antioxidant. Blood iscentrifuged at 1000 g for 10 mm; the supernatant is then centrifuged at10° C. and 1000 g for 5 min, followed by centrifugation at 15,000 g for10 min. This procedure removes all cellular debris, and a completelyclear plasma is obtained. Generally plasma is not stored but is used thesame day for LDL. isolation. The most common method for isolation of LDLis a two-step sequential ultracentrifugation with a total run durationof about 48 hr. LDL is prepared for oxidation experiments by a single20-hr run with a discontinuous density gradient. Plasma (up to 4 ml)adjusted with solid KBr to a density of 1.22 g/liter is layered on thebottom of a centrifuge tube (Beckman polyallomer tubes, total volume13.2 ml) and then overlaid by KBr density solutions of 1.08 (3 ml), 1.05(3 ml), and 1.00 g/liter (to fill the tube) containing 1 g/liter EDTA(pH 7.4). All density solutions are purged with nitrogen before use. Thetubes are centrifuged in a Beckman SW 41 Ti rotor at 40,000 rpm at 100for 20 hr. After centrifugation the main lipoproteins very low-densitylipoproteins (VLDL), LDL, and high-density lipoproteins (HDL) are wellseparated from each other, and the LDL band characterized by the yellowcolor due to the endogenous β-carotene, is collected by aspiration witha syringe and transferred into a polycarbonate tube.

[1641] Next, the cholesterol content of the isolated LDL sample isdetermined with the CHOD-PAP enzymatic test kit (Boehringer, Mannheim,Germany). When 4 ml normolipidemic plasma is centrifuged, the final LDLstock solution harvested from the ultracentrifugation has aconcentration of total cholesterol of about 1.6 to 2.2 mg/ml. Based onthe known composition of LDL the total cholesterol values can beconverted to LDL mass per milliliter (multiply cholesterol by the factor3.16) or LDL protein per milliliter (multiply total cholesterol by thefactor 0.63). It is also possible to determine the LDL concentration byprotein measurement. Next EDTA is from the LDL stock solution and theoxidation is conducted immediately after isolation of LDL. For storagethe LDL stock solution is sterile filtered through a 0.3 um filteradapted to a syringe into a sterile, evacuated glass vial andsubsequently purged with nitrogen (Techne Vial,Mallinckrodt-Diagnostica, Holland, or Behring, Marburg, Germany).

[1642] Removal of EDTA: Removal of EDTA and salt from the densitygradient from the LDL stock solution is conducted with prepacked columns(Econo-Pac 10DG, Bio-Rad, Richmond, Calif.) filled with Bio-Gel P6 asdesalting gel. The bed volume is 10 ml with a void volume of 3.3 ml, andthe total column volume is 30 ml. The gel is preconditioned by passing20 ml phosphate-buffered saline (PBS, 10 ml sodium phosphate buffer, pH7.4, containing 0.15 M sodium chloride) through the column.

[1643] A volume of 0.5 ml of the LDL stock solution is then applied tothe column. After the LDL solution has run into the gel, 2.5 ml PBS isapplied. The first 3 ml of eluate are discharged. The column is theneluted with 1 ml PBS, and 1 ml EDTA-free LDL solution is collected in a1.5-ml Eppendorf vial. The vial is immediately made oxygen-free bynitrogen gassing and transferred to a refrigerator. An aliquot isremoved to determine again the concentration by the CHOD-PAP method. TheLDL solution can be rather unstable at this stage, depending on thedonor, and therefore the time elapsed between desalting and the finaloxidation experiment should not exceed 60 mm.

[1644] Thiobarbituric Acid-Reactive Substances as Index of Low-DensityLipoprotein Oxidation: The preferred assay in LDL oxidation studies,both in presence and absence of cells, is the determination ofthiobarbituric acid (TBA)-reactive substances (TBARS) by one of the TBAassays developed for lipid peroxidation studies. The basal value ofTBARS in freshly prepared LDL samples is usually low (0.5 to 3 nm/mg LDLprotein) or undetectable. In LDL oxidized for about 24 hr with cells orCU²⁺ ions, the TBARS are in the range of 30 to 100 nmol/mg protein. Incopper-stimulated oxidation, formation of TBARS shows a lag phase ofabout 40-150 min depending on the LDL, temperature, medium, and Cu2+concentration; during this lag phase TBARS do not increase. Thereafter,TBARS rapidly increase for about 1-2 hr to a plateau value. On prolongedincubation TBARS remain more or less constant or increase slightly. Thereported time course studies for TBARS in cell-mediated oxidationsuggest that oxidation proceeds similarly to Cu²⁺ oxidation, with a lagphase followed by a rapid increase to a plateau level. In this context,it should be noted that most researchers only determine TBARS as an endpoint after about 24 hr incubation, when LDL has reached a final stageof oxidation.

[1645] Assays used for measurement of TBARS: Specifically, 100 ul of anLDL preparation (50 μg LDL cholesterin or 150 μg protein) is added to 1ml of 20% trichloroacetic acid (TCA). Following precipitation, 1 ml of1% thiobarbituric acid (TBA) is added, and the mixture is heated 45 minat 95°, cooled on ice, and centrifuged (20 min at 1000 g). TBARS arethen determined by measuring the absorbance at 532 nm or the emissionfluorescence at 553 nm (excitation 515 nm). Calibration is done with amalonaldehyde standard prepared from tetramethoxypropane.

[1646] The second assay is typically as follows: LDL (25 μg protein) ismixed with 1.5 ml of 20% TCA and 1.5 ml of 0.67% TBA. After heating at1000 for 30 mm, TBARS are determined fluorimetrically at an emissionwavelength of 553 nm with excitation at 515 nm. The sensitlvity wasreported to be 0.1 nmol TBARS/assay. This is equivalent to 4 nmolTBARS/mg protein. Haberland a al. determined the malonaldehyde-LDLadduct using a TBA assay. The malondialdehyde (MDA-treated LDL wasprecipitated with heparin-manganese, the supernatant was dischargedafter centrifugation, and the precipitate was washed withheparin-manganese prior to the TBA test.

[1647] Minimally modified LDL (MM-LDL) is prepared by dialyzing nativeLDL against 9 uM FeSO₄ in PBS for 72 h at 4° C. The electrophoreticmobility increased 1.1 to 1.2 versus native LDL. Mildly oxidized LDL wasalso obtained by (UV+copper/EDTA)-mediated oxidation under mildconditions: LDL solution (2 mg of apo/B/ml containing 2 umol/literCuSO4) was irradiated for 2 h. as a thin film (5 mm) in an open beakerplaced 10 cm under the UV-C source (HNS 30W OFR Osram UV-C tube, 1_(max)254 nm, 0.5 milliwatt/cm² determined using a Scientech thermopile Model360001), under the standard conditions. At the end of the irradiation,aliquots were taken up for analyses and oxidized LDL (200 μg of apoB/mlunder standard conditions or at the indicated concentration) wereimmediately incorporated in the culture medium.

[1648] Acetylation of LDL is performed with excess acetic anhydride.Endotoxin contamination in oxyLDL is measured with the coagulationLimulas amebocyte lysate assay using a commercially available kit(E-TOXATE, Sigma Chemical Co.).

[1649] Induction of Apoptosis by oxyLDL: Incubation of HUVEC with oxLDLfor 18 hours induced DNA fragmentation in a concentration-dependentmanner with maximal effects at 10 μg/mL. In contrast, native LDL did notinduce apoptosis in the concentration range tested. The induction ofapoptosis by oxyLDL is confirmed by demonstrating DNA fragmentationthrough agarose gel electrophoresis. LDH release did not increase ³10μg/mL oxLDL (105±11% compared with control cells) excluding theinduction of necrosis.

[1650] Detection of Fas and FasL Expression on Endothelial Cells: 90%confluent HAECs and HUVECs were incubated with oxyLDL (150 g protein/ml)or L-a-palmitoyl lysophosphatidyleholine (LPC, 45 uM, Sigma ChemicalCo.) at 37° C., 5% CO2 for 13 h, and detached from the culture platewith 0.5% EDTA. To determine FasL expression, endothelial cells areincubated with an anti-FasL antibody (C-20, Santa Cruz Biotechnology,Santa Cruz, Calif.) or with rabbit IgG followed by a FITC-conjugatedantibody against rabbit Ig (Biosource, Camarillo, Calif.). To determineFas expression, endothelial cells were incubated with an FITC-conjugatedanti-Fas monoclonal antibody (clone UBZ. Immunotech. Wemtbrook. Me,) oran FITC.conjugated mouse IgG. Immunofluorescence staining was analyzedby FACS (fluorescence-activated cell sorter) (Becton Dickinson. MountainView, Calif.).

[1651] Detection of DNA fragmentation by agarose gel electrophoresis:IIUVECs (10⁶) were incubated in the presence or absence of native LDL(300 μg protein/ml). oxyLDL (300 μg protein/ml). LPS (100 endotoxinU/ml), or a neutralizing anti-FasL antibody (i0 μg/ml, 4H9,MBL, Nagoya.Japan) for 36 h. Attached cells and floating cells were combined andlysed in 0.33 ml of lysis buffer (10 mM Tris-HCl. pH 8.0, 1 mM EDTA,0.2% Triton X-100) followed by incubation with 0.1 mg/ml RNAase A for 1h at 37° C. and 0.2 mg/mi proteinase K for 3 h at 50° C.Ethanol-precipitated DNA was resuspende in TE buffer, fractionated on1.5% agarose gel in 1×TBE buffer, and stained with ethidium bromide.

[1652] Detection of DNA fragmentation by TdT-mediated dUTP nick-endlabeling (TUNEL): 70% confluent HUVECs are incubated in the presence orabsence of OxLDL (300 μg protein/ml), a neutralizing anti-FasL antibody(10 μg/m 4H9). or an agonistic anti-Fas antibody (0.5 μg/ml CH11, MBL)for 16 h at 37° C., 5% CO₂. Attached cells harvested by trypsinizationand floating cells are combined, fixed in 4% paraformaidehyde,permeabilized in 0.1% Triton X-100, 0.1% sodium citrate, and incubatedwith TUNEL solution (Boehringer Mannheim. Indianapolis. Ind.) in theabsence or in the presence of terminal deoxynucleotidyl transferase.After washing in PBS, fluorescence intensity was analyzed by FACS.

[1653] Cell viability assay: HAECs or HUVECs are cultured in a 96-wellplate at 80% confluency and incubated in the presence or absence ofoxyLDL (300 μg protein/ml), LPC-C 16:0 (55 uM). a neutralizing anti-FasLantibody (10 μg/ml. 4H9). or an agonistic anti-Fas antibody (0.5 μg/ml.CH11) for 18 h. Cell viability is measured by means of MTT(dimethyithiazol-diphenyltetrazolium bromide) assay and percentage ofcell death was calculated as 100×(1−viability of treated endothelialcells/viability of untreated endothelial cells).

[1654] Cell Viability Assay and Reagents-Human umbilical veinendothelial cells (HUVECs) are isolated and cultured in endotheialgrowth medium (SCM; Clonetics, San Diego, Calif.). HUVECs cultured in a96-well plate at 80% confluency are incubated with oyxLDL or LPC atindicated doses for 16 h. Cell viability is measured by means of MTT(3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay.

EXAMPLE 50 Preparation of Vesicles Expressing Recombinant Membrane BoundSuperantigens Using the Yeast sec6 Mutant

[1655] The superantigen cDNA, or oxyLDL receptor, apoprotein verotoxinor other polypeptide given herein corresponding to the cDNA for proteinexpression in yeast is used. The length of the 5′-untranslated region isminimized. Expression of a cDNA in the sec6-4 yeast mutant is bestcontrolled and may be maximized with an inducible promoter. The GAL1promoter is preferred. The pYES2 expression vector (InVitrogen, SanDiego, Calif.) contains the GALI promoter followed by a multiple cloningsite. Other commonly used inducible promoters include themetallothionein CUPI promoter, which is tightly controlled by copper;promoters activated in response to heat shock, which are of particularinterest for expression in the temperature-sensitive sec6-4 mutant andthe PH05 promoter, which is derepressed at low phosphate concentrations.Introduction of the plasmid into yeast cells is accomplished either byelectroporation or LiCi-mediated transformation. Isolation oftransformants requires selection yeast that are ura3 auxotrophs are ableto grow on media lacking uracil when they contain the pYES2 expressionvector that contains the wild-type URA3 gene. Other selectable markersinclude enzymes in the adenine, histidine, leucine, lysine, andtryptophan biosynthetic pathways. The superantigen cDNAs are cloned intothe pYES2 expression vector and selected for transformants on plateswith synthetic complete (SC) medium lacking uracil but containing 2%raffinose as the carbon source (SC-Ura raff medium). Single colonies areisolated and grown overnight to saturation in 2 ml of SC-Ura raff mediumat 250 with constant shaking in 2% raffinose instead of glucose. In asubsequent step the yeast are switched to medium containing galactose asthe carbon source as the GAL1 promoter initiates gene expression onlywhen galactose is the predominant carbon source. The 2-ml starterculture in SC-Ura raff medium is added to a 1-liter culture of the samegrowth medium and incubated at 25° with constant shaking. When thesecultures reach an OD600 (optical density at a wavelength of 600 nm) ofabout 1.0 (usually about 12 hr), the cultures are centrifuged at 4000 gat 4° for 5 min, resuspended in 4 liters of SC-Ura gal induction medium(containing 2% galactose instead of 2% raffinose as the carbon source),and shifted to 37° for 2-3 hr to induce protein expression in the sec6vesicles.

[1656] Following growth at 37°, the cells are collected bycentrifugation at 4000 g at 40 for 5 mm and washed once in ice-coldwater. Pellets are resuspended in an absolute minimum volume of waterand quick frozen in liquid nitrogen. Cultures may then be storedindefinitely at −70°. Thawed cultures are resuspended to a finalconcentration of 50 OD618˜units/ml (e.g., a 1 -liter culture at 0D600=1.0 is resuspended in 20 ml) in 10 mM dithiothreitol (DTF) and 100 mMTris-CI, pH 9.4. The resuspended culture is shaken gently at roomtemperature for 10 min. This step increases the efficiency ofspheroplast lysis at a later step by reducing disulfide bonds in theyeast cell wall. We then collect the cells by centrifugation at 4000 gat 40 for 5 mm and resuspend them in spheroplast buffer to a finalconcentration of 50 OD600 units/ml. Spheroplast buffer consists of 1.4 Msorbitol, 50 mM K2HPO4, pH 7.5, 10 mM NaN3, and 40 mM 2-mercaptoethanol.Spheroplasts are generated by digesting the cell wall with lyticase (orzymolyase) for 45 min at 37°, The amount of bacterially expressed,recombinant lyticase needed to form spheroplasts is determinedempirically; after 45 min the OD₆₀₀ of a 10-ul aliquot of the yeastsuspension diluted into 1 ml of 0.1% sodium dodecyl sulfate (SDS) shouldbe ˜20% of the OD₆₀₀of the initial dilution measured at 0 min. Thespheroplasts are then harvested at 3000 g for 5 min at 4°, and the cellsare resuspended gently with a pipette or Teflon rod in spheroplastbuffer containing 10 mM MnCl₂ to a final concentration of 50 OD₆₀₀units/ml. Concanavalin A (Sigma, St. Louis, Mo.) is then added to afinal concentration of 0.78 to 1.25 mg/ml and incubated with rotation orgentle shaking at 40 for 15-30 mm. A concanavalin A stock solution (25mg/ml) is prepared in spheroplast buffer containing 1 mM MnCl2 and 1 mMCaCl₂ and is frozen in 1-mi aliquots. Lectin-coated spheroplasts areharvested at 3000 g for 5 mm at 40 and then resuspended in lysis bufferto a final concentration of 60-70 0D₆₀₀ units/mi. The suspension ishomogenized using the loose pestle of a Dounce homogenizer and 30-40strokes of the pestle at 40 (or on ice). Lysis bufferconsists of 0.8 Msorbitol, 10 mM triethanolamine (TEA), and 1 mM EDTA. The pH is adjustedto 7.2 with acetic acid or TEA.

[1657] Unlysed cells, cell debris, mitochondria, and nuclei are pelletedat 20,000 g for 10 min at 4° C. The supernatant is removed with apipette and centrifuged at 144,000 g for 1 hr at 40 to pellet thesecretory vesicles. The supernatant is decanted carefully and the pelletis resuspended in either lysis buffer or another buffer containingosmotic support.

EXAMPLE 51 Use of Antisense Oligonucleotides to Inactivate ITIMS InVitro and In Vivo

[1658] Antisense agents inhibit specific gene expression by exploitinghybridization of complementary nucleic acids, resulting in decreasedmRNA stability, or through blocking mRNA processing, transport ortranslation. An RNA or single stranded DNA molecule that iscomplementary to the mRNA of ITIM is introduced into cells in vitro. Theantisense molecule forms base-pairs with the mRNA, thus blocks someessential step in translation.

[1659] Antisense oligonucleotides can be delivered by plasmids or assmall single-stranded oligonucleotides. Modifications of thesestrategies, include inducible antisense vectors, antisense retroviralvectors, and a variety of oligonucleotide modifications to facilitatedelivery and enhance efficacy are known in the art.

[1660] Expression vectors are constructed to produce high levels ofantisense RNA in transfected cells. Antisense nucleic acids can beintroduced into cells of the immune system using syntheticsingle-stranded DNA oligonucleotides. When short oligonucleotides arecomplementary to the sequence around the translational initiation site(the AUG codon) of mRNA in the cells, they hybridize to the mRNA andprevent initiation of translation. Chemically modifying theoligonucleotides can greatly increase the efficiency with which theyenter cells and their stability once inside.

[1661] Principle of the Method: Genetically engineered plasmid-basedantisense methods, expression vectors which generate RNAs containingsequences complementary to key regions of specific genes are used. Theseantisense expression vectors include prokaryotic or eukaryoticselectable markers that allow identification of transfectants, apromoter that controls the expression of the antisense RNA, an antisensesequence which is complementary to a bindable region in the target genesequence (5′ untranslated region and/or translation initiation regionspreferred); and a stabilizing sequences to assure stability of theantisense RNA product.

[1662] Optimal gene replacement protocols include both inhibition of theendogenous gene and overexpression of a selected gene.Oligonucleotide-based antisense methods use synthetic single-strandedoligonucleotides which may range from simple deoxyribonucleotides tomore complex molecules containing base modifications and/or covalentmodifications which enhance delivery, uptake or antisense effect.

[1663] Materials and Reagents: Antisense expression vectors are obtainedfrom the laboratories where they were developed or are constructed bycombining the key elements described above. These include neomycin as aselectable marker so transfectants can be isolated employing Dulbecco'smodified Eagle's medium with ¹⁰% calf serum and the appropriateconcentration of the antibiotic G418 (Bethesda Research Laboratories.Bethesda, Md.). G418 permits selection of stable transformants.

[1664] Pure oligonucleotide preparations and nuclease-free cultureconditions are utilized. Antisense oligonucleotides are synthesized bysolid state methods and purified by chromatography or gel purification(or obtained from commercial sources). Following purification, theoligonucleotides are lyophilized two to five times in sterile distilledwater to remove volatile components. Both unmodified and modifiedoligonucleotides are suspended in 10 mM HEPES(N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid)-buffered saline atpH 7.4. The pH of these oligonucleotide solutions is checked to avoidthe toxicity of nonphysiologic pH. Nuclease-free culture conditions areemployed.

[1665] Design and Construction of Antisense Expression Vectors: Forinhibition of ITIM expression in mammalian cells, a prototype antisenseexpression vector is used that allows cloning of antisense and control“sense” gene fragments between the metal-inducible metallothioneinpromoter and β-globin sequences which are added to provide stability tothe antisense sequences. We have also constructed vectors which differonly in the promoter region because this region is readily excised by5′EcoRI and 3′ HindIII sites. One series of vectors is regulated by thesteroid-inducible mouse mammary tumor virus (MMTV) promoter. and anotherseries of vectors is regulated by the constitutive Rous sarcoma virusLTR. Antisense retroviral vectors are constructed which are compatiblewith the plasmid expression vectors: the promoter cartridge is flankedby unique 5′ XhoI and 3′ HindIII sites, and the antisense cartridge isflanked by unique 5′ HindIII and 3′ BamHI sites. Because the targetsequence for the 84-bp antisense vector resides entirely within the5′-untranslated region, this antisense vector inhibits the endogenousITIM gene.

[1666] Antisense-resistant expression vectors are constructed by cloningthe HaeII-EcoRI fragment of human ITIM cDNA into a Moloney LTR-regulatedexpression vector removing sequences complementary to the 84-bpantisense ITIM construct. In order to demonstrate which domains of theITIM gene contribute to cellular inhibition, ITIM C-terminal deletionmutants are constructed by linker insertion of an in-frame terminationcodon. All of these mutant ITIM plasmids are subcloned into expressionvectors for expression in cultured fibroblasts (Moloney-LTR promoter) orF9 embryonal carcinoma cells (Rous sarcoma virus LTR promoter).

[1667] Production of Stable Transformants Expressing Antisense RNAi:Stable transformants expressing both the antisense-resistant vector andthe anti-ITIM RNA construct are obtained by cotransfection of bothplasmids using the following protocol which generally results in 20-50transformants per 20-50 mm² tissue culture dish.

[1668] Day 1. 1.2 million cultured mouse fibroblasts (NIH 3T3,BALBc/3T3) are plated per 100-mm² tissue culture plates in 10 ml ofDulbecco's minimal Eagle's medium supplemented with 10% calf serum andallowed cells to attach overnight.

[1669] Day 2. Each plate is transfected by calcium-DNA coprecipitationwith 20 mg of cesium-banded plasmid DNA. For cotransfection studies, use2 mg of the antisense vector containing the Neo selectable marker and 18mg of the antisense-resistant expression vector which lacks thisselectable marker. This results in expression of both plasmids in moststable transformants. A DNA-calcium solution is prepared by mixing inthe following order: 20 mg of plasmid DNA dissolved in TE (10 mM Tris,pH 7.5. 1 mM EDTA), 50 ml of 2.5 M CaCl₂ and TE to make 500 ml total.This solution is added slowly to 500 ml of 40 mM HEPES-buffered saline(pH 7.2) and is then mixed by bubble aeration to form an opalescentsolution. After a 30-mm incubation at 25°, the entire: 1-mlcoprecipitate is added to the plate. After incubating the DNAcoprecipitate with the cells for 4 hr. the media is aspirated, and thecells are treated with 5% glycerol in 20 mM HEPES buffer for 3 min.After this glycerol shock, the cells are washed again withphosphate-buffered saline (PBS) and then refed with 10 ml of freshmedium containing serum.

[1670] Day 3. The media is aspirated and replaced with 10 ml ofDulbecco's minimal Eagle's medium supplemented with 10% calfserum and 1mg/ml of the antibiotic G418.

[1671] Day 5. The medium is aspirated (which by this time contains deadcells and cellular debris from G418-sensitive cells) and refed with 10ml of Dulbecco's minimal Eagle's medium supplemented with 10% calf serumand 1 mg/ml of the antibiotic G418.

[1672] Day 7. The medium is aspirated and refed with 10 ml of Dulbecco'sminimal Eagle's medium supplemented with 10% calf serum and 1 mg/ml ofthe antibiotic G418.

[1673] Days 9-12. Once individual clones are clearly apparent they canbe isolated with cloning rings and expanded in individual wells ofmicrotiter plates. Multiple clones should be studied for eachcombination of antisense vector and antisense-resistant rescue plasmid.

[1674] Cotransfectant clones are analyzed to quantitate the extent ofantisense inhibition of the ITIM gene, the level of expression of theantisense-resistant transfected gene, and the effect of target geneinhibition on cellular reactivity to lipid antigens or SAgs presentedalone or in the context of cell bound or soluble MHC or CD1 molecules.

[1675] Assays to quantitate reactivity and analyze antisense inhibitionin the presence and absence of antisense RNA are carried out as follows:[³H]Thymidine incorporation of stable transfectants and controls ismeasured after exposure to lipid antigens or SAgs in the context of MHCor CD1 either cell bound (or immobilized) or in soluble form. Control“sense” plasmids are similarly treated and tested. In addition, rescuewith a wild-type antisense-resistant gene should overcome the antisenseinhibition, provide additional assurance that the growth inhibition isactually due to specific inhibition of the ITIM gene.

[1676] Antisense inhibition is quantitated by measuring levels of eithertarget gene m RNA or protein. Nuclease protection assays provide anexcellent method for quantitation of antisense effect. Cells from thestable transformant clones are placed in DMEM with 0.5% calf serum for48 hr prior to steroid treatment and/or serum restimulation. RNA isisolated from cells by the guanidinium thiocyanate method and the totalRNA from 2×10⁶ cells is hybridized with 3×10⁶ cpm of the labeled RNAprobe for 16 hr at 45°. Samples are then treated with 2 mg/ml of RNase Aand 4 U/ml of RNase Ti for 30 min at 25° and are deproteinized bysequential treatment with proteinase K and phenol-chloroform followed;by ethanol precipitation. and electrophoresis on an 8% denaturingpolyacrylamide gel.

[1677] Antisense Inhibition with Anti-ITIM Oligonucleotides:Oligonucleotides target regions of the ITIM mRNA or SLAP RNA which areavailable for hybridization i.e., a region that is unbound by proteinand free of secondary structure. Sequences within the 5′-untranslatedregion or translation initiation region are employed for design ofantisense oligonucleotides. The length of oligonucleotides employed forantisense inhibition varies between 12 and 20 nucleotides. A meltingtemperature™ of 50-55° is optimal for specific inhibition of targetgenes. Tm for short oligonucleotides is best determined by a formula inwhich G and C residues equal 40 and A and T residues equal 20. OptimalITIM antisense oligonucleotides range from 14 to 19 nucleotides.

[1678] Determination of Oligonucleotide Stability: Oligonucleotides are5′ end labeled with polynucleotide kinase and [³²P]ATP and are thenadded to culture media and serum (plus unlabeled oligonucleotide toachieve a final oligonucleotide concentration of 5 mM: for a 15-mer thisis approximately 20 mg/ml). Aliquots are removed periodically andresolved on denaturing 20% acrylamide gels.

[1679] Detection of Intracellular Duplexes: To detect intracellularduplex, 20 mg of oligonucleotide is 5′ end labeled oligonucleotides with20 mCi of [³²P]ATP to achieve a specific activity of 50 million cpm/mg.After incubation for 4 hr. unincorporated oligonucleotides are removedby washing the cells three times with HEPES-buffered saline prewarmed to37° (to prevent the melting of duplexes). The cells are then lysed in100 ml of Nonidet P-40 lysis buffer (10 mM Tris, pH 7.5. 10 mM NaCl. 3mM MgCl2, 0.05% Nonidet P-4t)) containing 0.5% sodium dodecyl sulfate,100 mg of proteinase K per ml. and a 10,000-fold excess of unlabeledoligomer (as the carrier). Following phenol/chloroform extraction andethanol precipitation. a S1 nuclease protection assay is performed at37° and the products are analyzed on a 20% denaturing acrylamide gel(containing 42 g of urea of 100 ml). To demonstrate that the duplex isintracellular and not an artifact of RNA isolation, an “add-back”control is performed in which the measured amount of cell-associatedradioactivity is added with carrier (excess unlabeled oligonucleotide)to a lysate of cells that were previously unexposed to oligonucleotide.

[1680] To confirm that antisense oligonucleotide effects are dueentirely to target gene inhibition, and not merely toxicity due tounexpected effects of double-stranded RNA, reversal experiments areperformed employing an excess of anticomplementary (“sense”)oligonucleotide. If an effect is due to hybridization of antisenseoligonucleotide sequences to specific gene target sequences, thenaddition of an excess of sense oligonucleotide reverses the observedeffects on cell growth or gene expression but expression of theantisense rescue gene restores the ITIM function. This provides a usefulspecificity control for the antisense RNA and suggests that the observedresults are due to inhibition of endogenous gene expression.

[1681] Oligonucleotide preparations can have toxic effects on cells,thus anti-sense oligonucleotide these experiments employ multiplecontrols to assure that the observed results are not merely due totoxicity. First, the target gene is shown to be inhibited by analyzinglevels of mRNA and/or protein. Secondly, the demonstration thatoligonucleotides with sufficient sequence dyshomology are able toprohibit duplex formation. and third that the addition of ananticomplementary oligonucleotide can reverse the antisenseoligonucleotide effects by hybridization competition.

EXAMPLE 52 ITIM and SLAP Gene Knockout Mice

[1682] Gene Targeting and Generation of Mutant Mice

[1683] Nucleic acids encoding the ITIM of the IR_(TAA), IR_(IDA) andIR_(SAg) and SLAP are deleted in vitro using homologous recombinationand insertional mutagenesis. With this approach, mice are produced thathave mutations in any gene for which one has a cDNA or genomic clone.Stable cultured lines of totipotent cells, such as from mouse embryos orES (ES) cell lines, grown and manipulated in tissue culture areintroduced into blastocyst-stage mouse embryos and can participate inthe development of all tissues of the embryo, including the germ line.ES cells genetically altered in culture (e.g., whether by addition ormutation of genes) are used to produce strains or lines of mice thatcarry and transmit this genetic alteration. Cells which have undergonehomologous recombination are rendered positively selectable byintroduction of a marker gene that can be inserted into any cloned DNAmolecule. Negatively selectable markers are introduced at one end of thetransfecting DNA molecule to allow selection against random insertionsin parallel with the selection for the positively selectable marker.With these and other technical advances it is now feasible to mutate acloned ITIM gene by site-specific mutagenesis in ES cells and therebygenerate a an inbred mouse line with the desired mutation and functionaldeficiency.

[1684] Gene Targeting Vectors

[1685] Homologous recombination frequency is higher when the genetargeting vector is isogenic with the ES cells and also increasesmarkedly with increasing sequence homology. With this in mind, genomicclones are isolated that contain 3-5 kb of sequence homology on eachside of the intended ITIM or SLAP mutation (except for the case whereinPCR is used to screen for homologous recombination).

[1686] To make an ITIM or SLAP knockout or null mutation, a deletion ismade in the ITIM gene, followed by insertion of a positive selectablemarker. Insertion alone is more likely to allow some expression of thegene by aberrant transcription and/or translation. The ITIM or SLAPmutation is made near the 5′ end of the gene and includes deletion ofsome of the sequence encoding, the mature protein. An effective strategyis to delete the ATG start codon and the signal sequence if they fall ina single exon. If the transcription start site is known, this is alsodeleted. Such deletion of control elements for transcription,translation and secretion increases the probabilitty that expression ofthe desired protein will be completely ablated. The positive selectablemarker is then inserted in place of the deleted segment.

[1687] The positive selectable marker is one that confers antibioticresistance (to G418, hygromycin B or an equivalent antibiotic) byreplacing the bacterial control sequences of the neomycin resistance(neo) or hygromycin resistance (hyg) genes, respectively, witheukaryotic control sequences. Both markers are expressed at many loci inES cells when controlled by the mouse phosphoglycerate kinase (PGK)promoter and polyadenylation signal. The engineered pMCI promoter of thegene encoding hypoxanthine-guanine phosphoribosyltransferase (HGPRT) isalso used successfully in ES cells for gene targeting.

[1688] To promote targeted versus random integration of the targetingvector, a positive/negative selection procedure is used which utilizes anegative selectable marker at one or both ends of the targeting vector.The negative marker is lost during homologous recombination but retainedduring random integration. The negative selectable marker utilized isthe herpes simplex virus thymidine kinase gene (HSV-TK), which renderscells sensitive to ganciclovir. HSV-TK works well under control ofeither the pMCI or the PGK promoter. The same promoters are used forboth positive and negative selectable markers. To minimize random breaksbetween them, the distance between shoulld be is less than about 5 kb.The targeting vector has a unique restriction site that permitslinearization before transfer of the DNA into cells. Several vectorswhich have eight-cutter sites (e.g., the NotI restriction site) in theirpolylinkers are used for these constructs. Screening for targeted cellsis by genomic Southern blotting using a probe that is outside thetargeting vector or by by PCR.

[1689] Specific Methodology: The methods disclosed herein are allconventional in the art and are described, for example, in George E L etal., Methods in Enzymol.245: 386-420. (1994) which are herebyincorporated by reference in their entirety.

[1690] ES Cells: The ES cell lines: CC1.2, CCE, D32 ABI, and JI areuseful and are tested for germ line transmission by implanting intoblastocysts of a mouse strain having a different coat color genotype. EScell lines are derived from the 129/Sv mouse strain which carries thewild type agouti gene (brown color). Injection of these ES cells intoC57BL/6 (strain with black coat color) blastocysts produces chimericoffpsring with a mix of black and agouti coat color. When the ES cellscontribute to the-germ line, agouti progeny are born (because agouti isdominant to black).

[1691] ES cells are are maintained in culture on mouse embryonicfibroblast (MEF) feeder cells and/or in the presence of a growthfactor—leukemia inhibitory factor (LIF)—and are used at low passagenumbers to maintain totipotency and avoid differentiation. The MEFfeeders are prepared from embryos of an appropriate transgenic mousestrain that carries genes for resistance to the positive selection drug(e.g., G418/neomycin, hygromycin).

[1692] Reagents for ES Cell Culture: ES cell medium is a supplementedDulbecco's modified Eagle's medium (DMEM) to which has been addedL-glutamine, and sodium pyruvate and HEPES buffer; the pH is maintaineddat 7.5. For use, the medium is supplemented with the followingadditives: fresh glutamine, fetal bovine serum (FBS, finalconcentration, 15% v/v), nonessential amino acids, 2-mercaptoethanol andLIF (final concentration, 1000 U/ml) of LIF. All these components arecommercially available, e.g., from GIBCO-BRL (Life Technologies, Inc.)Feeder cell medium is the above DMEM-based medium without the additives,but supplemented with FBS (10% v/v). Phosphate-buffered saline (PBS):0.8% NaCl, 0.02% KCl, 0.02% KH₂PO₄, 0.115% NaHPO₄ (all w/v) at pH 7.4.Ethylenediaminetetraacetic acid (EDTA) is at a final concentratin of 0.2g/liter in PBS, pH 7.2. Trypsin-EDTA: 40 ml of EDTA, with a 2.5% (w/v)concenttation of trypsin. HEPES-buffered saline (HBS): HBS: 25 mM HEPES,134 mM NaCl, 5 mM KCl, 0.7 mM Na₂PO₄, pH 7.1.

[1693] Preparation of MEF Feeder Layers; Preparation of Primary MEF: ForG418-resistant feeders, a transgenic mouse line containing the neo geneis used. Similarly, if hygromycin is used for selection, feeder cellshave the hyg gene.

[1694] 1. Fibroblasts are obtained by aseptically removing embryos froma 14-day pregnant mouse into PBS. One embryo at a time is dissected toremovve fetal membranes, placenta, the head and soft tissue. Each embryois placed in a separate petri dish and minced with two scalpels or finecurved scissors. Trypsin-EDTA (fresh, 2 ml) is added to each dish andincubated at 37° C. for 5 min. Then, the reaction is stopped by additionof 8 ml of feeder cell medium to each dish, and and the contents aretransferred to a conical centrifuge tube and allowed to settle for 2min.

[1695] 2. The cell-containing supernatant of each tube is poured intoone 10-cm diameter culture dish. Fresh medium is added 24 hr, and thecultures are maintained in a standard tissue culture incubator for about1-2 days until the cells are confluent.

[1696] 3. The cells of each dish are then split into ten dishes each (10cm) and again cultured until confluent (about 2 days).

[1697] 4. The cells or each dish are trypsinized and resuspended in 1 mlof ice-cold freezing medium (feeder cell medium, but with 20% (vlv) FBSandd 10% (vlv) dimethyl sulfoxide (DMSO). Aliquots of 1 ml per vial arefrozen and stored at −80° for 24 hr. For long-term storage, vials arekept at −135° C. or in liquid nitrogen.

[1698] Feeder Layers for ES Cells: Mitotically inactivated (3,000 radsof y-irradiation) MEFs at 2.5×10⁶ cells/dish (“dish” refers to a 10 cmdiameter culture dish unless otherwise specified) are used. To achieve auniform monolayer of feeders, plates are coated with 0.1% (w/v) gelatinfor at least 5 min prior to plating cells. Feeders are either pre-platedor co-plated with ES cells. If the cellsa e to be co-plated, or if nomedium change is planned before addition of ES cells, it is important towash the feeder cells out of DMSO by centrifugation because ES cells aresensitive to this compound.

[1699] Routine Culture of ES Cells: ES cells are cultured MEFs (whichare at a density of 2.5×10⁶/dish) for up to 10 days. ES cells are platedat relatively high density, 3×10⁶ to 10⁷/dish. They grow rapidly, anddivide every 18-24 hr. ES cell cultures are fed daily with ES cellmedium. The cells need to be passaged about every third day, and requirea second feeding on the day before passage. Generally, higher viabilityis achieved if the cells are re-fed 2-3 hr prior to any trypsinization(for passaging or freezing).

[1700] Passaging ES Cells

[1701] 1. After rinsing the monolayers, 2 ml of prewarmed trypsin-EDTAis added to each dish which is held at 37° C. for about 5 min at whichtime the cells visibly detach. Trypsin action is stopped by adding 2 mlof ES medium. Cells are dispersed thoroughly with a Pasteur pipette andtransferred to a conical centrifge tube with an additional 6 ml ofmedium. The cells are spun for 2 min at 500 rpm. This treatment isimportant because ES cells do not grow well in the presence of trypsin.

[1702] 2. The supernatant is removed by aspiration and replaced with 2ml of fresh ES medium, in which the cells are dispersed by gentlepipetting (to avoid bubbles, etc.) with a Pasteur pipette 20 times.Thorough dispersal of ES cells is important as aggregates are morelikely to differentiate.

[1703] 3. For passage, ES cells are usually split 1:6, although higherdensity may be desired. Even before confluence ha been reached, culturescan be split in order to disperse the ES cell colonies to prevent theirdifferentiation.

[1704] Freezing and Thawing ES Cells.: The ES cells are re-fed 2-3 hrbefore trypsinization (see above) for freezing. They are frozen at highdensity; the contents of one confluent dish geneeally becomes six 1-mlvials. ES cells are especially sensitive to DMSO toxicity, so exposureis minimized by resuspending cells (with thorough dispersal) in ice-coldmedium without DMSO first, and then adding an equal volume freezingmedium which contains 2× DMSO. Vials are frozen slowly for 24 hr at−80°, then store at either −135° or in liquid nitrogen for long-termstorage.

[1705] For use, the ES cells are thawed rapidly in a 37° water bath andquickly transferred to ice. The cells are spun out of DMSO in about 10ml of ES medium, resuspended with thorough dispersal and are plated onfeeder cells as described.

[1706] Transfection of ES Cells by Electroporation

[1707] 1. Linear DNA is prepared cconventionally by phenol-chloroformextraction and ethanol precipitation. Vacuum-dried DNA is resuspended insterile HBS. An aliquot is checked for degradation by comparingrestriction maps of linearized and intact plasmid DNA using multiplerestriction enzymes.

[1708] 2. Cells are split 1:6 as usual, 1 or 2 days prior toelectroporation. Cells are fed daily and again 2-3 hr prior toelectroporation. Cells are trypsinized as usual but, on dispersal ofcells, about 10 ml of medium is added and returned to the incubator for30 min. This pre-plating allows about 90% of feeder cells to reattach,further enriching ES cells. Cells are washed twice in HBS and an aliquotis counted.

[1709] 3. The cells are resuspended in ice-cold HBS at 2.5×10⁷ cells/ml.Linearized DNA is added from a concentrated stock in sterile HBS to afinal concentration of 25 mg/ml, and the tubes are placed on ice for 10min. After mixing with a Pasteur pipette, cells plus DNA are transferredinto an electroporation cuvette (Bio-Rad, Richmond, Va.) and are kept onice for 10 min. Each electroporation cuvette holds 0.8 ml, equivalent toabout 2×10⁷ cells.

[1710] 4. Electroporation is carried out at 240 V, 500 mF with the GenePulser (Bio-Rad, Richmond, Va.) with a capacitance extender. The cellsare allowed to rest for 10 min at room temperature after which they areplated with, neomycin-resistant feeders (but no selection drugs) atabout 5-7×10⁶ ES cells/dish.

[1711] Selection of Targeted Clones: Beginning at 24-36 hrs afterelectroporation, the cells are fed daily with selection medium untilselection is complete (usually 7-8 days). Cell death is observed by 3-4days after drug addition. Selection Medium includes G418 (Geneticin;GIBCO-BRL) final concentration. The G418 is dissolved in HEPES buffer.The final concentration G418 is 200 mg/ml if PGK-neo is in the targetingconstruct. If pMCI-neo is in the construct, then a 150 mg/ml solution isused. For double selection, ganciclovir (Syntex Corp., Palo Alto,Calif.) is included at a 2 mM final concentration. An alternative togancyclovir is FIAU (Bristol Meyers Squibb, Wallingford, Conn.), whichis used at 0.2 mM.

[1712] Culture medium is replaced by selection medium beginning at 24-36hr of culture. One or two plates are grown in G418 medium only, and areused to calculate the number of neomycin-resistant colonies. Cells arefed daily. Cell death is visible at about 3-4 days of selection. Thecolony morphology is observed carefully. Some large colonies startflattening. Clones are picked routinely at day 7 or 8 of selectionbefore they start differentiating (but flattened colonies may stillcontain ES cells and can be picked). Plates are stained with Giemsa, andcolonies counted on day 10 of selection.

[1713] Other Selection Measures: The above procedures are suitable forthe commonly used schemes involving the neo gene as positive selectionmarker and the HSV-TK gene as the negative selection marker. If hyg,HGPRT- or puromycin-resistance genes are used, the feeders are modifiedaccordingly. One can either obtain a line of mice with the appropriatetransgene or use a feeder cell line just during the time of selection. Aline of STO cells doubly resistant to neomycin (G418) and hygromycin canalso be used. ES cells can, and should, be returned to feeder culturesat the time of picking the clones. Selection for HGPRT is in “HATmedium” which is standard medium supplemented with 0.1 mM hypoxanthine,0.8 mM aminopterin and 20 mM thymidine. Hygromycin selection utilizesconccentratios ranging from 100 to 150 mg/ml and should be tested todetermine the effective concentration.

[1714] Picking Drug-Resistant ES Cell Colonies: Clones are usuallypicked after 7 or 8 days of selection. Each clone is picked into anindividual well of a 96-well plate containing trypsin-EDTA. Eachtrypsinized colony is then split into a single well of duplicate 24-wellplates, so that 1 plate can be used for freezing, and the other for DNAextraction for Southern blot or PCR analysis. The transfer of cellsbetween 96-well plates and 24-well plates is accomplished with a12-channel pipettorwit tips in alternating channels. This allows thetransfer of clones from wells #1, 3, 5, 7, 9 and 11 oof a 96-well plateinto wells #1, 2, 3, 4, 5, and 6 of a 24-well plate.

[1715] Trysinization of Selected Clones

[1716] 1. Selection plates are fed 2-3 hr before picking colonies. Thenumber of colonies to be picked is estimated, and 24-well plates withabout 10⁵ MEF/well in 0.5 ml are prepared. Each colony is split in halfinto wells of duplicate 24-well plates.

[1717] 2. After washing once, trypsin-EDTA at 37° C. is placed in 1 rowof a 96-well plate (60 μl/well). Colonies are picked rapidly in groupsof 12 (one row), and the trypsin-EDTA is maintained at 37° C. Individualclones are picked by tearing each away from the surrounding feedercells, using a ripping/tearing motion, and drawing each gently into thepipette. Before approaching a colony, the pipette is partially filleedwith PBS from the plate to prevent sticking of the colony to the glass,using as little volume as is suffficient to prevent the colony fromsticking. Each clone is transferred to a well of the 96-well platecontaining trypsin-EDTA.

[1718] 3. Six pipette tips are connected to the multichannel pipettor(channels 1, 3, 5, 7, 9, and 11) and clones 1, 3, 5, 7, 9, and 11 aredispersed by pipetting up and down several times. The cell suspension in30 μl is transferred to the first row of one 24-well plate (prepared asabove) and the remaining 30 μl to the first row of the duplicate 24-wellplate. Clones 2, 4, 6, 8, 10, and 12 are dispersed and transferred in ananalogous manner. (Alternatively, the trypsin reaction is stopped afterpicking 12 colonies in the first row of the 96-well plate by adding 60μl complete medium and dispersing the cells. One can then pick the next12 colonies into the second row. This is most helpful when the selectionplates contain more than 12 colonies.) The cells are fed as soon aspossible and then daily with 0.5 ml of complete medium.

[1719] Freezing Expanded Colonies: One of the duplicate 24-well platesis used. Cells are frozen when many of the wells are subconfluent, evenif some wells are still sparse. This is important to ensure that themajority of the colonies have not grown too large and the cells begun todifferentiate. Cells are frozen, at this stage only, in mediumcontaining 10% (v/v) DMSO, fetal calf serum, and trypsin-EDTA. For thisrelatively short-term storage, the trypsin-EDTA does not appear to harmthe cells.

[1720] 1. The cells are fed 2-3 hr before freezing. 120 μl of anice-cold solution of 20% (v/v) DMSO and 80% (v/v) FBS (“ECS solution”)is placed in each Costar cluster tube and maintained on ice. The cellsare washed with warm PBS and trypsinized with 60 μl of pre-warmedtrypsin-EDTA for 5 min at 37° C. The plate is moved to ice, and eachwell receives 180 μl of ice-cold ECS. The cells are dispersed bypipetting up and down with six tips of the multichannel pipettor. Twofrozen vials of each clone are prepared by transferring 120 μl of thesuspension (clones 1-6) into one cluster tube (tubes 1, 3, 5, 7, 9, 11)and the remaining 120 μl into the neighboring tube in the same row(tubes 2, 4, 6, 8, 10, and 12). Thus, each row of clustered tubescorresponds to each row of the 24-well plates.

[1721] 2. To freeze the cells slowly, the cluster tube rack is wrappedin paper towels, placed at −20° for about 1 hr, then transferred to−70°. The cluster tube racks are maintained at −70° until positiveclones are identified.

[1722] DNA Isolation for Genomic Southern Blot Analysis

[1723] 1. Clones are grown until most are confluent. High-quality DNAcan be extracted even from overgrown clones. Wells are washed with PBS,200 μl of lysis buffer are added to each well, and incubated at 37° for30 min. Plates are stored frozen at this point. The lysis buffer:consists of 150 mM NaCl, 20 mM Tris (pH 7.5), 5 mM EDTA, 0.5% (w/v)sodium dodecyl sulfate (SDS), proteinase K (0.25 mg/ml).

[1724] 2. Lysates are transferred to 1.5-ml tubes. Racks are arranged tofit the format of a 24-well plate so that a 12-channel pipettor can beused to process the samples through all the steps.

[1725] 3. Lysate is extracted with an equal volume of phenol-chloroform;the mixture is vortexed, centrifuged and the organic phase is removedusing a 12-channel pipettor. The amterial is then extracted withchloroform-isoamyl alcohol. The aqueous phase is transferred to cleantubes and precipitated with 20 ml of 3M sodium acetate (pH 5.2) and 0.5ml ethanol, at −20° for 30 min.

[1726] 4. The pellets are resuspended in 10 mM Tris-HCl (pH 8.0), 1 mMEDTA (TE). The precipitation is repeated as described above, the pelletsare dried and resuspended in TE. To dissolve DNA, the solution is leftat room temperature for several hours or at 65° C. for 15 min. Aconfluent well from a 24-well plate yields approximately 10 μg ofgenomic DNA. One-half or one-third of each sample is digested withrestriction enzymes. To aid in complete digestion, a relatively large(100 ml) volume is used and allowed to proceed overnight. The digestscan then be precipitated or reduced in volume in a Speed-Vac (Savant,Hicksville, N.Y.) before loading on a gel.

[1727] Polymerase Chain Reaction Screen of ES Cell Clones.

[1728] An alternative to the Southern blotting screen described above isto use the PCR, which is done on pools of clones. Characterization of EScell clones by PCR in pools of 12 (blot analysis of PCR products isusually required) is performed as follows. Clones are maintained inculture during this quick initial screen. Positive pools are rescreenedas individual clones by PCR, and subsequently confirmed by genomic blotanalysis. A PCR primer is characterized that is within the targetingvector (usually in the positive selection marker) and another in genomicDNA outside the targeting vector. These primers are tested for theirlowest detection limit and extent of cross-homology. To ensure reliableamplification in small amounts of genomic DNA, the amplificationdistance is only about 1 kb. This limits the homology with the locus tobe targeted that is included in the targeting vector and thus. decreasestargeting frequency.

[1729] 1. Drug-resistant colonies are picked by the procedures describedabove, except allow colonies to settle overnight in 96-well dishes withfeeders. Each colony is split into two 96-well dishes by trypsinizationof the attached cells. One dish is for PCR analysis and the other isexpanded. Each dish has a feeder cells and 0.2 ml of medium. Using amulti-channel pipettor, routine trypsinization is performed using nomore than 50 ml of trypsin-EDTA. After thorough but gentle dispersal ofcells, 25 ml is transferred to each 96-well dish with 0.2 ml of mediumper well. The cells are still in dilute trypsin but can tolerate itovernight, which is enough time to settle. They are refed after 12 hr.

[1730] 2. One to 2 days after the initial split, the cells aretrypsinized from 1 of the duplicate 96-well dishes as usual, but do notadd medium with serum.

[1731] 3. Samples are combined by row (12 wells/pool) by dispersing intrypsin-EDTA, followed by a rinse with PBS. Microfuge the pools for 3min. to spin down the cells.

[1732] 4. All but 5 ml of supernatant is removed and the pelletresuspended in 50 ml of water and frozen on dry ice.

[1733] 5. Cells are thawed at 95° for 8 min and cool. 10 mg ofproteinase K (fresh aliquot) is added, incubated at 55° for 30 min. andincubated at 95° for 8 min. and cooled.

[1734] 6. Components for a standard 100-ml PCR reaction are added. 20 mlof each PCR reaction is run on an agarose gel and blot (variousquick-blot procedures are adequate). Probe with subclone of partialamplification product.

[1735] 7. The remainder of each clone is expanded by continued culturinguntil the results of pooled PCR analysis are complete and fed daily.

[1736] 8. Once the PCR results are in, all clones are individuallypassed within each positive pool to a 24-well plate with feeders.One-half of each sample is saved for PCR analysis as an individualclone.

[1737] 9. The cells are fed daily. After 3 days, once the PCR analysisof clones is complete, each positive clone is placed on a 35- or 60-mmplate with feeders to expand for freezing (see below). Feed daily.

[1738] Thawing and Expanding Targeted Clones: For each positive clone,thaw one of the cluster tubes rapidly in a 37° water bath, then cool onice. Do not spin. Plate on a 35-mm plate with feeder cells (4×10⁵ feedercells per plate) in 2 ml of complete medium. Feed after 12 hr. At thispoint, cells are cultured in normal medium without G418; it has beenreported that cells maintained in G418 have a lower capacity tocontribute to the germ line of injected embryos. Depending on thedensity of each clone, split up to 1:6. Freeze as soon as possible insmall aliquots. Freeze enough aliquots for one aliquot per day ofblastocyst injection. A subconfluent 60-mm plate can be frozen as eight250 ml aliquots, in 90% (v/v) FCS and 10% (v/v) DMSO. Such an aliquotcan be plated directly after thawing into one or two 35-mm plates withfeeder cells, 2 days before injection.

[1739] Further Characterization of Targeted Clones: Positive clonesdetected by the above procedures are retested using several different:restriction enzyme digestions to ensure appropriate insertion of theselectable marker into the locus; both flanks of the insertion arechecked. To determine if targeted clones have any additional insertionsof the targeting vector, DNA is tested with a probe containing the neogene. Only a single band at the expected size is detected after longexposure. A further simple check of the condition of targeted cells isto count chromosomes:

[1740] 1. A rapidly growing culture is treated with Colcemid (0.06mg/ml) for 4 hr.

[1741] 2. Cells are trypsinized, washed in PBS, and combined with washedcells from culture supernatant.

[1742] 3. The cells are resuspended in 10 ml of 0.56% (w/v) KCl,incubated at room temperature for 8 min. and spun at 300 rpm for 2 min.at room temperature.

[1743] 4. All but 100 ml of supernatant is removed and the cellsresuspended. 10 ml of fixative [3:1 (v/v), methanol-glacial acetic acid]is added dropwise and incubated for 10 min.

[1744] 5. The cells are spun and resuspended in 1 ml of fixative.

[1745] 6. The cells are dropped, from a height of 10 cm, onto glassslides (2-3 drops per slide, cleaned with ethanol and dried). After theliquid has spread to the edges of each slide, blow dry.

[1746] 7. After they are completely air dried, stain the slides for 15min. in 3% (w/v) Giemsa in PBS, wash in water and air dry.

[1747] 8. To count chromosomes is to photograph the slide on print filmso that counted chromosomes can be marked. At least 30 spreads/clone arecounted. At least 50% of the spreads have 40 chromosomes. Watch out forspreads with too many chromosomes as well as those lacking chromosomes.

[1748] Generation and Breeding of Chimeric Mice: The animal andequipment requirements and procedures for generating chimeric mice byinjection of ES cells into blastocysts is as follows. C57BL/6 mice areused as embryo donors and strain CD1 as foster mothers. Contribution ofES cells, which are derived from strain 129/Sv mice and have agouti coatcolor, is clearly evident in contrast to the black coat color of thedonor embryo.

[1749] The following procedure yields healthy ES cells as a single-cellsuspension. Two days prior to blastocyst injection, one of the frozenaliquots [90% (v/v) serum, 10% (v/v) DMSO] is thawed rapidly in a 37°water bath and plated directly into two 35-mm dishes with feeder cells(4×10⁵ feeders per plate). The medium is replaced the next morning,daily, and 2 hr before trypsinization. One plate is trypsinized at thebeginning of an injection session and the second about 3 hr later, infreshly prepared 0.25% (v/v) trypsin (GIBCO-BRL) 0.02% (v/v) EDTA in PBSat 37° for 5 min. Cells are gently dispersed after adding an equalvolume of ES cell medium and pellet. Cells gently resuspended (bydrawing them in and out of a Pasteur pipette 20 times) in 2 ml ofice-cold ES cell medium and kept on ice until transfer to the injectionchamber.

[1750] General guidelines for blastocyst injection: The procedurerequires an inverted microscope with interference-contrast orphase-contrast objectives. The magnification range required is ×40 to×200. The injection microscope is equipped with a cooling stage(blastocysts are more resilient to the injection procedure when they arekept at 5-10°). A pair of micromanipulators is used one for the holdingpipette and one for the injection pipette. The manipulators from LeitzInstruments are especially useful. Flow in each pipette is regulated byhand, using a micrometer to adjust a Hamilton syringe. To obtain donorembryos, a stereo dissecting microscope (total magnification, ×20) isrequired for flushing blastocysts from the uterus and collecting themfor culture. Blastocysts are cultured in a tissue culture dish, inmicrodrops of medium under light-weight paraffin oil, in 5% C02 in airat 37° During an injection session, several small batches of blastocystsare transferred from culture to the injection chamber with trypsinizedES cells. The easiest blastocysts to inject are those that have expandedfully but have not yet hatched from the zona pellucida. Loading ES cellsinto the injection pipette is done with care so as not to damage thecells. The injection pipette is just large enough to accommodate the EScells, but not much larger, because damage to the blastocyst would bemore likely to occur. Each blastocyst is injected with 15-20 ES cells.After injection, the blastocysts are returned to culture for at least 1hr before surgical transfer to a pseudopregnant female. The surgery isperformed on the bench with general anesthesia, using a stereodissecting microscope. Each female receives 10-12 blastocysts;uninjected blastocysts are transferred along with injected ones, to helpprevent complications in the pregnancy due to small litter size.

[1751] Chimeric animals are monitored by coat color. The 129/Sv-derivedES cells give rise to an agouti (brown) coat whereas the recipientC57BL/6 embryos produce black coat color. Desirable chimeras have a highproportion of brown coat. To determine if targeted ES cells havecontributed to the germ line of chimeric mice, male chimeras are bredwith C57BL/6 females. Agouti pups are the result of ES cell-derivedsperm. If the targeted mutation is heterozygous viable, then one-half ofthe agouti pups will carry the targeted allele. However, if none of thechimeras derived from a given ES cell clone has high coat colorchimerism then germ line transmission is unlikely. Discard chimeras ifnone of the first 60 pups contains the targeted allele and, because mostES clones give chimeras of which around 50% give germ line transmission,we would discard a clone if none of the first 5 or 6 chimeras givesagouti pups.

[1752] It is necessary to obtain germ line transmission from at leasttwo independently derived ES cell clones in order to ensure that anyphenotype of the resulting mice arises from the targeted mutation andnot from some other mutation that occurred during handling of the EScells.

[1753] Genotyping Mice: Tail Blots and PCR Analysis

[1754] Genomic DNA, isolated from tail biopsies, is analyzed by eitherSouthern blot or PCR. Generally, mice must be at least 3 weeks old totolerate the general anesthesia used in cutting the tail. With a freshrazor blade, cut a 1-cm length from the tip of the tail and cauterizethe remaining tail with a soldering iron. DNA is isolated by asimplified procedure that is amenable to large numbers of DNA samples.Transfer the tail biopsy to a 1.5-ml tube that contains 0.5 ml of taillysis buffer: 100 mM Tris-HCl (pH 8.5), 5 mM EDTA, 0.2% (w/v) SDS. 200mM NaCl, proteinase K (100 mg/ml) (Boehringer Mannheim, Indianapolis,Ind.). Continuously rotate the samples overnight at 55° and Vortex thetubes, and spin down hairs and tissue debris in a microfuge for 5-10min. Transfer the supernatant to a fresh tube containing 0.5 ml of2-propanol and mix thoroughly. Recover the precipitate with a pipettetip and transfer to a fresh tube containing 100 ml of 10 mM Tris-HCl,0.1 mM EDTA, pH 7.5. Make certain that DNA is dissolved by incubation at37° with intermittent vortexing.

[1755] To analyze tail DNA by Southern blot, a single-copy probe thathybridizes with wild-type and targeted alleles is required. Generallysuch a probe has already been characterized for use during screening fortargeted ES cells. Generally 15 ml of DNA prepared as above issufficient for one lane of a Southern blot. Restriction digestion shouldbe carried out in a final volume of at least 50 ml, with at least 40units of enzyme and in the presence of bovine serum albumin (BSA; 0.1mg/ml) and 4 mM spermicide. Digestions usually require several hours toovernight for completion. If the DNA does not digest well, aphenol-chloroform extraction followed by reprecipitation is likely tohelp. A variety of standard blotting procedures suitable for genomic DNAcan be used. Capillary blotting to a nylon membrane, ultraviolet (UV)cross-linking, and hybridization in sodium phosphate and SDS at 65° workwell.

[1756] As an alternative, PCR amplification of unpurified tail DNA usingappropriate PCR primers can be used. Tail (5-10 mm) is added to 0.4 mlof PCR tail buffer 150 mM KCl, 10 mM Tris-HCl (pH 8.3), 2.5 mM MgCl₂,gelatin (0.1 mg/ml), 0.45% (wlv) Nonidet P-40 (NP-40), 0.45% (w/v) Tween20]. Incubate overnight at 55° with shaking and with addition of two 25ml aliquots of proteinase K (10 mg/ml) added at an interval of severalhours. Heat at 95° for 10 min to denature residual proteins, cool toroom temperature, and spin. Five microliters of each DNA sample shouldgive definitive ethidium bromide signals after PCR amplification withprimers for each allele.

[1757] To analyze DNA by PCR, primer pairs are designed that indicatethe presence of the wild-type allele versus the targeted allele. This isaccomplished as follows: First, both alleles are assayed by a set ofthree primers, one of which is within the neo gene, as described inPolymerase Chain Reaction Screen of ES Cell Clones, above.Alternatively, separate primer pairs are used to assay the alleles:onepair within the neo gene, and the other pair in the wild-type gene. Inboth these strategies, designing primers that yield amplificationproducts of different sizes allows detection of both alleles in a singlereaction tube and gel lane. Care is taken not to contaminate reagentswith amplification products. Organization of reagents into single-usealiquots is highly recommended.

[1758] Generation of Homozygotes: To determine if homozygous mutantanimals are viable, heterozygous crosses are performed and all pupsgenotyped at weaning. The expected 1:2:1 ratio of genotypes, or lack ofit, becomes evident in three to four litters. However, genotype ratiosare kept for all heterozygous crosses performed. Ideally, mice derivedfrom two or three independent targeted clones are available.Heterozygous crosses is carried out for each targeted clone, as well asbetween clones. The interclonal crosses show that any phenotype is theresult of the targeted mutation rather than some other mutationoccurring in the ES cell clone.

[1759] For a homozygous viable mutation, the next job is to show thatthe targeted mutation is truly a null allele. Procedures will depend onthe gene/protein of interest. This has been performed quite thoroughlyfor the tenascin-deficient mice and for P-selectin-deficient mice atboth the mRNA and protein level.

EXAMPLE 53 Animal Models of Infectious Diseases for TestingSuperantigen-Lip-IDA Conjugates, Anti-Sense Oligonucleotides, GeneKnockout Mice, Immune Cells Deleted of Inhibitory IR_(IDA) and/or ITIMsor ITAMs

[1760] Abbreviations: IR_(IDA)=Inhibitory Receptor for Lipid-BasedInfectious Disease Associated Antigens, Lip-IDA=Lipid-Based InfectiousDisease Associated Antigens, IR_(sAg)=Inhibitory Receptor forSuperantigens, IR_(TAA)=Inhibitory Receptors for Lipid-Based TumorAssociated Antigens.

[1761] Tuberculosis:

[1762] C57BL/6 Bg/Bg mice, which are NK cell-deficient, are infectedwith any one of a nontuberculous species of mycobacteria: M. aviumComplex, M. kansasil, M. simiac, M. malmoense or M. genavense. Same-sexmice aged 5-7 weeks are allowed to acclimate for 1 week in the facilitybefore being used. They are housed in microisolator units (Lab Products,Maywood, N.J.) and are randomly distributed six to a group.

[1763] Inoculum and Infection Process: Primary cultures of MAC (M.kansasii or other mycobacteria) to be used for infection are obtainedfrom clinical isolates of patients with disseminated MAC infection, orthe American Type Culture Collection (ATCC). ATCC 49602 (serotype 1)strain LPR and MAC 101 (provided by Lowell Young, California PacificMedical Center Research Institute, San Francisco, Calif.) are used.Organisms are grown in modified 7H 10 broth (7H 10 agar formulation withagar and malachite green omitted), pH 6.6, with 10% (vol./vol.)Middlebrook oleic-acid-albumin-dextrose-catalase (OADC) enrichment(Difco Laboratories, Detroit, Mich.) and 0.05% (vol./vol.) Tween 80(Sigma, St Louis, Mo.). Broth cultures are started from one transparent,smooth, flat colony (SmT) grown on an agar plate. The culture tube isplaced in an orbital shaker and incubated at 37° C. for 3-5 days.Culture suspensions are predorminately (>95%) of the smooth,transparent, and flat (SmT) phenotype which is more virulent and moreresistant to antimycobacterial agents than the smooth, domed, opaque(SmD) or rough phenotypes. After incubation, the culture is diluted in7H10 broth to a concentration of 10 Klett units/ml (Manostatcolorimeter, Manostat, New York, N.Y.) or approximately 5×10⁷ cfu/ml.The inoculum is titrated in triplicate on 7H10 agar plates (Difco)supplemented with 5% (vol./vol.). Middlebrook OADC enrichment. Platesare taped with Blenderm® (3M, St Paul, Minn.), incubated for 2-3 weeksat 37° C., and then counted to determine the precise inoculum.

[1764] Treatment Schedule and Controls

[1765] 1. Aerosol infection at low doses: The preferred model is toexpose mice to a very low inoculum of bacilli using an aerosolgeneration chamber. After uptake of about 50 bacilli in the lungs theinfection grows progressively at first and is then curtailed around 20days. Laboratory strains of mycobacteria such as Erdman attain 4-5 logsin the lungs by this time. More virulent strains such as CSU93(Tennessee outbreak) and strabn W (New York) can grow to between 6 and 7logs in this time.

[1766] 2. Intravenous infection at high doses: The high-dose intravenousmodel is also employed. Most of the inoculum is taken up by phagocytesin the spleen and liver and only 1-2% can be detected in the lungs. Theorganisms grow progressively for 10-15 days in the spleen, and to alesser extent in the liver, until acquired immunity is generated,resulting in a “chronic” disease pattern. Thus, the genetic constructunder test is given soon after inoculation or after the disease hasbecome chronic. This more closely resembles the human condition as thepatient is probably at this stage before initial diagnosis.

[1767] The inoculum is injected iv in a 0.2 ml volume using a 0.5 mlsyringe with an attached 28 g needle to deliver a total of approximately10⁷ cfu/mouse. Each experiment consists of an early control group(sacrificed 1 week postinfection at the initiation of therapy) and alate control group (sacrificed at the end of therapy), neither of whichreceives any treatment. One treatment group consists of a drug withknown to activity (e.g., azithromycin or clarithromycin). Treatment isstarted 7 days postinfection and is generally continued for 10 days insuccession. In extended therapy experiments, treatment is given daily(Mon-Fri) for 4 or more weeks.

[1768] Mice are weighed at the beginning and end of each trial, andaveraged by group. Mice are evaluated daily, and changes in appearanceor behaviour are noted. In general, infected mice appear outwardly well,and continue to gain weight with these infections. There have been someexceptions where the infected control animals have succumbed to theinfection. Untreated mice develop splenomegaly, hepatomegaly (withvisible lesions), and enlarged lungs. Splenomegaly, although minimal, isevident at 1 week postinfection (average weight 0.12-0.14 g) andcontinues to increase for the duration of the trial (average weight0.65-0.80 g at 18-20 days postinfection). After 1 week of infection, theusual organ bacterial counts in the lungs of early control mice(10^(4.5)) is significantly less than that in the spleen (10⁷). The latecontrol mice (19 days postinfection) average 10⁶ and 10^(7.8) cfu intheir lungs and spleens respectively.

[1769] At the completion of the trial (2 days after the end of thetreatment phase), mice are euthanized. Their spleens and right lungs areaseptically removed. Spleens are weighed. Each organ is placed in agrinding assembly containing normal saline and Tween 80 and each sampleis ground and allowed to sit for 15 min to allow for settling ofaerosols. The tops of the grinding assemblies are removed and an aliquotis removed. Dilutions are made to the appropriate concentrations forplating using tubes containing double-distilled H₂O (to promote redblood cell lysis) with saline-Tween 80.

[1770] Spleen weights are used to estimate dilutions for the plating ofthe homogenate. Large spleens, such as those belonging to members of thelate control group, are heavily infected and need to be diluted. Thelungs are less infected than the spleens in this model and usuallyrequire 10-100 times less dilution. Each organ is plated at threedifferent dilutions on 7H10 agar (Difco) plates containing 5% OADCenrichment. Plates are incubated for 2-3 weeks and counted to determineviable cell counts, which are expressed as counts/organ.

[1771] Key Parameters to Monitor Infection and Response to Treatment:Mice show some or all of the following signs of infection: a hunchedposture with or without difficulty ambulating, isolation from the restof the group in their cage, labored breathing, and shivering, lack ofeating and/or drinking and diarrhea.

[1772] Upon introduction of a new agent, mice are monitored initiallyand during the first hour for any indications of pain or discomfort,allergic reaction or swelling. During the first 3 days of therapy, themice are monitored at least twice daily to note their generalappearance. Any injection site is inspected for swelling or irritation.The general condition of the animals is carefully noted. Mice having anacute reaction are euthanized immediately.

[1773] Therapeutic Regimen and Treatment Schedule: Therapy is begun 7days postinfection. Determination of the spleen and lung viablebacterial cell counts is done several days after the completion oftherapy. Although daily treatment for 10 days allows for differentiationof relative activities, longer treatment periods (4-12 weeks) are usefulto characterize efficacy.

[1774] Administration of the therapeutic constructs is started on day 20(when the first animal becomes DTH-positive) and is continued 2-3 timeweekly for four weeks. Bacterial loads are determined at 35 and 50 days.Isoniazid (25 mg/kg/day) is given as the positive control drug in eachtrial.

[1775] Outcome: Treated animals show elimination and/or reduction ofviable organisms in the spleens and lungs or spleen weights of therapygroups in comparison to those in the spleens and lungs of the control(infected, but untreated) groups. These differences are statisticallysignificant using the Wilcoxin rank test or other statistical methodknown in the art. Additional comparison is made between the therapygroups and the group given a standard therapy such as clarithromycin orazithromycin.

[1776] Leishmaniasis: Infection Procedure: BALB/c mice are infected ivto give the quickest and most reproducible infection in the liver,spleeen and bone marrow. The inoculum of parasites, either amastigote orpromastigotes, is loaded into a 1 ml syringe fitted with a 23 g needle.For experimental infection, an inoculum of 10⁷ amastigotes in a volumeof 0.1 to 0.2 ml is injected intravenously (or in hamsters, by theintracardiac route). This will produce a microscopically detectableinfection in the liver of mice and liver and spleen of hamster after 1week of infection. This level is suitable for tests of the therapeuticconstructs desccribed herein.

[1777] Administration of Therapeutic Constructs: Therapeutic constructs[David: what are they?] are administered to mice by a variety of routes(s.c., ip. and p.o.) and for some formulations i.v. administration bythe tail vein is required. In the mouse model, treatment is bestevaluated against an established infection on days 7-11 post-infection.If a lower infection inoculum is used, tests can be carried out on days14-18 post-infection. In the commonly used BALB/c mouse the infection inthe liver increases linearly until days 21-28 following infection by 10⁷amastigotes or promastigotes; after this point the liver infectionbecomes chronic and eventually cure. The spleen infection, althoughmicroscopically detectable from week 1, is fully established after theweek 4. If the infection is left untreated for several weeks prior totreatment, chronic granulomatous infection is established which has beenshown to be less sensitive to standard drugs.

[1778] In an alternative approach in mice,, treatment with therapeuticconstructs is started immediately after infection. A 5-day course oftreatment is sufficient to determine relative potencies of theconstructs.

[1779] Outcome: In the early stages of infection VL [David: what is VL?]in mice presents no obvious external symptoms. Extra mice or hamstersare infected and sacrificed prior to administering the conjugates tocheck that the infection is established. Microscopic examination ofstained slides prepared from the liver and/or spleen of will indicatewhether the inoculum was satisfactory and that infection has beenestablished. The appearance of hamsters does change in the later stagesof infection. The most noticeable features are weight loss and dullingof the hair. Occasional hamsters may develop ascites. These clinicalchanges in the untreated controls are contrasted with changes in thetreated population. The treated groups show none of these changes in thecourse of therapy.

[1780] At the end of treatment the mice are weighed to give anestimation of drug toxicity. The livers and spleens are removed fromfreshly sacrificed animals and weighed. Smears are prepared from thelivers and spleens on microscope slides, fixed in methanol for 1 minuteand stained with Giemsa stain for 45 minutes. The number of parasitesper 500 liver cells and/or spleen cells is determined microscopicallyfor each experimental animal. This figure is multiplied by total organweight (mg) and this figure, the Leishman-Donovan unit (LDU) is used asthe basis for calculating the difference in parasite load betweentreated and untreated animals. Treated animals show complete eliminationor reduction in the number of parasites/500 liver and/or spleen cells.The difference in parasite loaded between the treated and control groupsis statistically significant using the Wilcoxon rank test. The activityof novel compounds is compared with that of the standard antimonialdrugs and expressed as a therapeutic ratio.

[1781]Trypanosoma cruzi {Chagas Disease): Chagas disease has beenobserved in different mammal species. Several animal models have beenused experimentally—mice, hamsters, dogs, rats, rabbits and monkeys. Thecourse of the T cruzi infection varies widely between these species,depending upon the host and parasite strains used, the route ofinoculation and the size of the inoculum.

[1782] Mouse Model: The mouse model is cheaper, easy to work with and itcan produce both the acute and the chronic phases of the disease.Various mice strains differ markedly in their resistance to T cruzi.More resistant strains might providea good model for the chronicdisease. At this stage, the murine model of Chagas' disease is used inexperimental therapy. Several strains of mice have been used in thismodel: Swiss, weight 18-20 g, female; Balb/c, 8-10 weeks, female; albino[what is albino? it's a mutation and coat color, not a strain!!], weight18-20 g male. Preliminary experiments are performed to determine theoptimal parasite inocula to insure infection. The parasites aremaintained by serial passage through female C3II/IIe mice, a resistantstrain. Mice with parasitemia are bled into heparinized (1000 UA) PBS(50:50) and cryopreserved.

[1783] Hamster model: Hamsters (non-isogenic Syrian hamsters,Mesocricetus auratus, male/female) are infected with T cruzi. During theacute phase an inflammatory reaction is observed characterized bymononuclear and polymorphous leukocyte infiltration of variable degreein the majority of tissues and organs. In the chronic phase the samekind of lesions can be observed, but the inflammatory process is lesssevere and characterized by mononuclear infiltration in the myocardium(Ramirez L E, et al., Rev Soc Bras Med Trop, 1994 27:163-916). Theauthors noted high levels of parasitemia in the beginning of theinfection, which varied with the strain used.

[1784] Parasite Strain & Stages of Disease: Different parasitic strainsbehave quite differently in experimental Chagas' disease with regard tocharacteristics such as the course of infection, the degree ofparasitemia, tissue tropisms, histopathological changes and mortality.Several strains of T cruzi have been used in different animal model andinclude Y Ernane, Benedito and Vicentina. Strains of T. cruzi, fromdifferent geographical areas, had previously been characterized intovarious types according to their infectivity rate and tropism in mice.The classification includes the following:

[1785] 1. Type I, characterized by a rapid course of infection in mice,high levels of parasitemia and mortality around the 9^(th) and 10^(th)day of infection, with predominance of slender forms and macrophagetropism during the acute phase of the infection.

[1786] 2. Type II shows increasing parasitemia from the 12^(th) to the20^(th) day of infection, low mortality rate, predominance of broadforms of the parasite and myocardial tropism.

[1787] 3. Type III shows a slow development of parasitemia that reachesa high level 20-30 days after inoculation, low mortality andpredominance of parasitism in skeletal muscles.

[1788] Inoculation & Infection Process: The inoculum range is usuallyfrom 10³ to 10⁷ trypomastigotes (obtained from infected animals) or2-4×10³ metacyclic trypomastigotes (obtained from triatomid bugs).Acceptable inoculation routes are intraperitoneal and conjunctival. Miceweighing. 18-20 g are inoculated by the intraperitoneal route with5×10⁴-1×10⁵ trypomastigotes which produces a homogeneous infection.Daily trypanosome counts provide the following pattern for theparasitemia: parasites appear from the 4^(th) or 5^(th) day afterinoculation, their number decreases markedly on the 6^(th) day,increases until the 7^(th) or 8^(th) day, and decreases again around the9^(th) day. From the 10^(th) day onwards the pattern of parasitemia isirregular. Most infected animals die in the period from the 5^(th) tothe 20^(th) day after inoculation; the highest mortality rates areobserved around the 15^(th) day. Mortality rates are about the same forboth sexes and only a small number of infected animals will outlive 40days.

[1789] The administration of constructs begins on the day afterinoculation and doses corresponding to about one-fifth of the LD₅₀ aregiven for 10 consecutive days. On the 5^(th) day after inoculation thenumber of parasites in 5mm³ of blood is determined. On the 8^(th) day,when the number of parasites in the inoculated animals is generallyhigher, a new count is performed.

[1790] Outcome: The best initial criteria for therapeutic activity inthe experimental Chagas' disease is mortality and parasitemia.Parasitemia is usually high with little variation, depending on thestrain used and follow-up is done by daily fresh blood examination. Theblood is collected and the parasites counted in a Neubauer's chamber.The acute phase of the infection is followed by a chronic stage in whichparasites are reduced to submicroscopic level, then indirect laboratorymethods are used, such as subinoculation, xenodiagnosis and serologicaltechniques. The polymerase chain reaction has been used as acomplementary criterion for therapeutic activity in the chronic stage ofexperimental Chagas disease.

[1791] The following techniques are used to establish reliable criteriafor cure in the mouse model of Chagas' disease:

[1792] 1. Fresh blood examination: a drop of blood from the tail iscarefully examined in a Neubauer chamber daily or every other day.

[1793] 2. Blood subinoculation: mice are killed about 1 or 2 monthsafter treatment and 0.4-0.6 ml of citrated blood, collected from thesevered axillary artery, is inoculated intraperitoneally intosusceptible mice. From the 5^(th) day of inoculation, fresh bloodexaminations are performed daily or every other day for a period of atleast 6 weeks.

[1794] 3. Blood culture: blood from treated animals is inoculated intoNoeller's culture medium and culture examined for at least 30 days afterinoculation.

[1795] 4. Xenodiagnosis: 1 or 2 months after treatment, mice areanesthetized and 4 triatomine nymphae are allowed to feed on them. After45-50 days, the bugs are carefully examined for trypanosomes

[1796] 5. Histological examination: histological sections of the heartof treated animals are stained with hematoxylin and eosin and examined.

[1797] 6. Re-inoculation: some of the treated animals are reinoculatedat different times after treatment with about 4000 blood parasitic formsper gram of body weight; daily counts of trypanosomes are performed sothat a new acute phase of the disease can be detected.

[1798] Treated animals show 80-100% cures and prolonged survivalassociated with elimination or reduction in parasitemia compared tountreated control groups. The differences are statistically significantusing the Wilcoxon rank test or other non-parametric or parametricstatistics known in the art.

[1799] Antisense Oligonucleotides and IR_(IDA), ITIM, ITAM or SLAP GeneKnockout to Delete or Inactivate IR_(IDA)s and/or ITIMs, ITAMS or SLAP

[1800] In a representative ex vivo protocol, using the murine TB,Leishmania and trypanosome models described above, an antisenseoligonucleotide at a dose of 0.01-1 mg corresponding to the codingregion of IR_(IDA) ITIM or SLAP is added to autologous T cells inculture. The uptake of the oligonucleotides by the cells is confirmedusing oligonucleotides labeled at the 5′end with fluoresceinisothiocyanate (FITC). To check for inactivation of the the IR and ITIMsor SLAP by the oligonucleotides, Western blot quantitation is carriedout on the lysed T cells. Expression of the IR_(IDA) genes and theirrespective ITIM genes is downregulated by >95%.

[1801] In a parallel experiment, T cell are deleted of the gene encodingthe IR_(IDA) and their respective ITIM or SLAP by homologousrecombination with a mutant gene. The knockout T cells and theantisense-treated T cells are expanded in IL-2 for 24-72 hours,harvested and reinfused into mice with established tuberculosis,leishmaniasis or trypansomiasis. Optionally the T cells are exposed toLip-IDAs for 24 hours before the addition of IL-2. The animals aresacrificed at the end of the experiment and assayed for residual diseaseas deseribed above. Results are statistically assessed as given aboveand show that the adoptively transferred knockout T cells induce greaterthann 95% reduction in the number of residual organisms.

[1802] In a representative in vivo experiment, mice are inoculated withorganisms. The antisense phosphorothioate oligodeoxynucleotidecorresponding to an antisense-sensitive part of the IR_(IDA) genes andof their respective ITIM genes and a control antisense construct (thesame nucleeotide composition but with a jumbled sequence) are used.

[1803] A single dose of ITIM or SLAP antisense or control antisense (1mg per 0.1 ml saline per mouse) or saline (0.1 ml per mouse) is injecteds.c. into the right flank of mice once the disease is established in theabove models At each indicated time, the animals from the control andantisense-treated groups are killed and residual organisms assayed asdescribed above.

[1804] The uptake of ITIM or SLAP antisense in the tissues is assessedby photoaffinity labelling followed by immunoprecipitation of ITIM orSLAP as follows: The tissues are homogenized with a Teflon/glasshomogenizer in ice-cold buffer 10 (Tris-HCl, pH 7.4, 20 mM; NaCl, 100mM; NP-40, 1%; sodium deoxycholate, 0.5%; MgCl, 5 mM; pepstatin, 0.1 mM;antipain, 0.1 mM; chymostatin, 0.1 mM; leupeptin, 0.2 mM; aprotinin, 0.4mg ml; and soybean trypsin inhibitor, 0.5 mg ml; filtered through a0.45-pm pore size membrane), and centrifuged for 5 min in an Eppendorfmicrofuge at 4° C. The amount of ITIM in organs is determined byphotoaffinity labelling with ⁸N-3-[³²P]cAMP followed byimmunoprecipitation with anti-ITIM antibodies.

[1805] In a second representative in vivo experiment, IR_(IDA)/ITIM orSLAP knockout mice are prepared by the above methods. They are theninoculated with organisms as above. On day three following injection,the mice are immunized with 0.1-1 mg of Lip-IDAs alone or conjugated toSAgs (see Section 51 and 55) in CFA. If unconjugated Lip-IDA is given,SEB (0.01-0.1 mg) is administered i.p. 2-3 times per week for threeweeks. Separate groups of mice are treated with Lip-IDAs or SAgs aloneor with CFA or saline or without either Lip-IDAs or SAg respectively.The mice are sacrificed on day 21 and the number of residual organismsassayed as above.

[1806] Alternatively, the antisense oligonucleotides complementatry toIR_(IDA) ITIM or SLAP (0.01-1 mg/mouse) are injected intravenously intomice with established disease. Lip-IDA and SAg or conjugates thereof(Sections 51 and 55) are given on the same schedule as immediatelyabove. Infectious burden is assayed at the end of experiments in bothtreated and untreated groups as indicated above. Results show >95%reduction in infectious burden in treated animals compared to controls.

EXAMPLE 54 Animal Tumor Models: Antisense Oligonucleotides and GeneKnockout Mice and Cells Deleted of Inhibitory Receptors for Lipid-BasedTumor Associated Antigens (IR_(TTA)) and/or Inhibitory Receptors forSuperantigens (IR_(SAg)) and/or their ITIMs and/or ITAMs and/or SLAP

[1807] In a representative ex vivo protocol, using the MCA 205/207murine sarcoma model in C57BL/6 mice (see Examples 15-16), antisenseoligonucleotide 0.01-1 mg corresponding to the coding region ofIR_(TAA)/IR_(SAg) ITIM or SLAP are added to autologous T cells in tissueculture. The uptake or the oligonucleotides by the cells is confirmedusing oligonucleotides labeled at the 5′end with fluoresceinisothiocyanate (FITC). To check for inactivation of the the IR and ITIMsor SLAP by the oligonucleotides, Western blot quantitation is carriedout on the lysed T cells. The IR_(TAA) and IRSA genes and theirrespective ITIM genes are downregulated by >95%. In a parallelexperiments, T cell are deleted of the gene encoding their IR_(TAA) orIR_(SAg) genes and their respective ITIM SLAP genes by homologousrecombination with a mutant gene. The knockout T cells and theanti-sense treated T cells are expanded in IL-2 for 24-72 hours,harvested and reinfused into C57BL/6 mice with established pulmonarymetastases as described in Example 16. Optionally the T cells areexposed to lipid-based TAAs or SAg or SAg transfected tumor cells or SAgtransfected tumor cell/dendritic cell fusion cells (Example 25-26) for24 hours before the addition of IL-2. The animals are sacrificed 21 dayslater and the number of pulmonary metastases counted. Results arestatistically assessed as given in Examples 15-16 and show that theadoptively transferred knockout T cells induce a >95% reduction in thenumber of pulmonary metastases.

[1808] In a representative in vivo experiment, MCA 205/207 tumor cells(1.5×10⁶) are inoculated s.c. into the left flank of C57/B1 mice. Theantisense phosphorothioate oligodeoxynucleotide corresponding to thecodons for IR_(TAA) and IR_(SAg) genes and their respective ITIM or SLAPgenes and control anti-sense (the same base composition as the antisensewith the sequence jumbled) are used. A single dose of ITIM or SLAPantisense or control antisense (1 mg per 0.1 ml saline per mouse) orsaline (0.1 ml per mouse) are injected s.c. into the right flank of micewhen tumour size reached 80-100 mg, 1 week after cell inoculation. Tumorvolumes are obtained from daily measurement of the longest and shortestdiameters and calculation by the formula, 4/30r³ wherer=(length+width)/4. At each indicated time, two animals from the controland antisense-treated groups are killed, and tumours removed, weighed,immediately frozen in liquid N, and kept frozen at −80° C. until used.

[1809] Photoaffinity labelling followed by immunoprecipitation of ITIMis carried out as follows: The tumors are homogenized with aTeflon/glass homogenizer in ice-cold buffer 10 (Tris-HCl, pH 7.4, 20 mM;NaCl, 100 mM; NP-40, 1%; sodium deoxycholate, 0.5%; MgCl, 5 mM;pepstatin, 0.1 mM; antipain, 0.1 mM; chymostatin, 0.1 mM; leupeptin, 0.2mM; aprotinin, 0.4 mg ml; and soybean trypsin inhibitor, 0.5 mg ml;filtered through a 0.45-pm pore size membrane), and centrifuged for 5min in an Eppendorf microfuge at 4° C. The supernatants are used astumour extracts.

[1810] The amount of ITIM or SLAPin tumours is determined byphotoaffinity labelling with 8-N3-[³²P]cAMP followed byimmunoprecipitation with the ITIM antibodies. In a representative invivo experiment, IR_(TAA)/ITIM Knockout C57BL/6 mice are prepared byabove methods. The MCA 205/207 tumor cells (2-3×10⁵) are then injectedintravenously to induce pulmonary metastases. On day three followinginjection, they are immunized with 0.1-1 mg of Lip-TAAs alone orconjugated to SAgs (Section 51 and 55) in CFA. If unconjugated Lip-TAAis given, SEB (0.01-0.1 mg) is administered i.p. 2-3 times per week forthree weeks. Separate groups of mice are treated with Lip-TAAs or SAgsalone or with CFA or saline or without either Lip-TAA or SAgrespectively. The mice are sacrificed on day 21 and the number ofmetastases in the lung are counted as given in Example 16.

[1811] Alternatively, the antisense oligonucleotides complementatry tothe IR_(TAA) or IR_(SAg) or ITIM or SLAP coding sequences (0.01-1mg/mouse) are injected intravenously into C57BL/6 mice with MCA 205/207tumors present subcutaneously ((at least 1 mm in diameter) or withpulmonary metastases established by injection of 2-3×10⁵ tumor cellsintravenously 3 days before. Lipid-based TAA and SAg or conjugatesthereof (Sections 51 and 55) are given on the same schedule asimmediately above. Tumor size is measured weekly in both treated anduntreated groups. The number of pulmonary metastases in the treated anduntreated groups is determined as given in Examples 16 and 21. Resultsare evaluated statistically as given in Examples 16 and 21 and show >95%reduction in tumor size and pulmonary metastases in treated animalscompared to controls.

EXAMPLE 55 Preparation of Lip-TAAs and Lip-IDAs of Bacterial, Fungal,Yeast, Parasitic, Mycobacterial, Invertebrate or Protozoan Origin

[1812] Isolation and Characterization of Glycosphingolipids: Thefollowing are procedures for obtaining purified glycosphingolipidantigens and lipid-based SAg receptors (digalactosylceramides) frombiological sources for use in treatment of cancer and infectiousdiseases. The major membrane bound glycosphinolipids useful fortreatment of cancer include the alpha mono-, di- andtri-galacotsylceramides in mammalian cells, phytosphingosines present inyeast, vertebrate and plant cells as the well established array of tumorassociated membrane antigen e.g., GD2 neuroblastoma associated antigens.It also includes the vast array of glycosphingolipid antigens associatedwith infectious diseases as given in Section 54.

[1813] The basis for extraction of glycosphingolipids from biologicalsources is their solubility in chloroform-methanol mixtures.Gangliosides (glycosphingolipids containing neuraminic acids) andglycosphingolipids with five or more carbohydrate residues are not onlysoluble in chloroform-methanol mixtures but also form molecularaggregates that are soluble in water. Glycosphingolipids with one tofour residues, form emulsions in water which is the basis for the Folchpartition (chloroform-methanol (2:1) with one-fifth volume of water ordilute KCl solution) in which gangliosides are partitioned into theupper water-methanol layer and neutral glycosphingolipids remain in thelower chloroform-methanol layer. Most glycosphingolipids are easilyextracted from tissue or other material with chloroform-methanol (2:1)but quantitative extraction of gangliosides requires more polarextraction mixtures such as chloroform-methanol (1:1) orchloroform-methanol (1:2). Metal ions also affect the distribution ofgangliosides in biphasic systems. Glycosphingolipids are separated froma total lipid extract by silicic acid column chromatography followed bythin-layer chromatography. Ion exchange cellulose (DEAE) columnchromatography is used to separate acidic compounds, such as sulfatideand gangliosides, from less acidic or nonpolar compounds.

[1814] Extraction and Partition: All solvents used in the followingprocedures are redistilled from glass to remove nonvolatile compounds.Chloroform is stabilized by the addition of methanol (afterdistillation) to a final concentration 0.25% (by volume).Chloroform-methanol and other mixed solvents are given as volume/volumeratios. A weighed portion of the tissue to be extracted is vigorouslyhomogenized with seven volumes of methanol (w/v) in a blender orhomogenizer. Fourteen volumes of chloroform are added and the mixturehomogenized again. The final solvent ratio is chloroform-methanol (2:1).The material is filtered with a Buchner funnel using an aspirator and acoarse-grade solvent-washed filter paper. The residue is reextractedwith 10 volumes (based on weight of the original material) ofchloroform-methanol (2:1). After filtration, the residue is extracted athird time with 5 volumes (v/v) of chloroform-methanol (1:1) orchloroform-methanol (1:2). The third extraction is only necessary forquantitative removal of gangliosides. The final combined extract isadjusted by addition of chloroform so that the final proportion ischloroform-methanol (2:1). A volume of 0.1 M KCl equivalent to one-fifththat of the final solvent extract is added, mixed vigorously, andallowed to stand at 4° overnight or until the layers are completelyseparated. If the volumes are small, the layers are separated bycentrifugation. The upper and lower layers are washed three times withtheoretical lower and upper phases, respectively, prepared by shaking amixture of 1 volume of chloroform-methanol (2:1) and 0.2 volume of 0.1MKCl in water and letting the phases separate. With large volumes ofcombined extracts, the solvents are evaporated in vacuo and the residueredissolved in a convenient volume of chloroform-methanol (2:1) for thepartition and washing steps described above.

[1815] The combined lower phases (original and washes) are collected andreduced in vacuo to a small volume with gentle warming (<50°) on arotary evaporator (fraction I). The combined upper aqueous phases aredialyzed against cold tap water for 24 hours and then lyophilyzed(fraction II). The lyophilyzed material, usually containing someinsoluble protein, is extracted with chloroform-methanol-water (10:5:1),filtered and reduced to a small volume on a rotary evaporator. FractionII generally contains only gangliosides and may be analyzed bythin-layer chromatography without further purification (see below).

[1816] Silicic Acid Column Chromatography: The lipids from thechloroform-methanol layer (fraction I) are fractionated into neutrallipids, glycolipids, and phospholipids on a column of Unisil silicicacid (Clarkson Chemical Co., Williamsport, Pa.). This procedure isuseful for glycolipids containing from one to four glycosyl residues andfor sulfatides. Inisil (20-40 g per gram of lipid) is activated at 80°for several hours and is slurried with chloroform as quickly as possibleafter removal from the oven and poured into a column. The adsorbent iswashed with about three bed volumes of chloroform or until it istranslucent. The column is not allowed to run dry. A 20 mg/ml solutionof the sample is applied in chloroform. Neutral lipids are eluted withabout five bed volumes of chloroform and then glycosphingolipids areeluted with 8 to 10 bed volumes of acetone-methanol (9:1). Phospholipidsare eluted with 5 bed volumes of methanol.

[1817] A different procedure has been used to isolate a particularglycolipid on a preparative scale, as in the purification of trihexosylceramide, gal (1→4) gal (1→4) glc-ceramide, from a kidney of a patientwith Fabry's disease (trihexosylceramidosis). A crude glycolipid andphospholipid mixture is obtained from fraction I by addition of 200 mlof ether and filtration of the resultant glycolipid-phospholipidprecipitate at room temperature. The glycolipid mixture (3 g in oneexperiment) is then subjected to mild alkali-catalyzed methanolysis. Asilicic acid column (400 g) is prepared in chloroform-methanol (19:1),and the sample applied in chloroform-methanol (19:1). The column iseluted successively with 1500 ml each of 12%, 14%, 16%, 20%, 30%, and50% methanol in chloroform. Fabry trihexosylceramide (1.5 g) is elutedas a pure compound in the 20% fraction. The 16% and 30% fractions alsocontain some (1 g) of the Fabry lipid mixed with other glycolipids. Asimilar mixture of glycolipids is fractionated on a silicic acid columnusing a continuous gradient from 5% to 50% methanol in chloroform.

[1818] Mild Alkali-Catalyzed Methanolysis: The glycolipid fraction fromthe silicic acid column is treated with mild base to removecontaminating phospholipids. This treatment does not affect glycolipidsor gangliosides unless they contain an O-acyl group. The followingquantities are used for 1-10 mg of glycolipid fraction. Add 1 ml ofchloroform and 1 ml of 0.6 N NaOH in methanol to the dry fraction andallow the mixture to react at room temperature for 1 hour. Then add 1.2ml of 0.5 N HCl in methanol, 1.7 ml of water, and 3.4 ml of chloroform,mix well, centrifuge, and remove the lower layer containing theglycolipids. Wash the lower layer three times with methanol:water (1:1)and then evaporate it to dryness in vacuo. If a ganglioside fraction isto be methanolyzed, the sample is treated in the same way except thatafter neutralization with methanolic HCl the sample is dried in vacuo,emulsified in water and dialyzed against tap water at 4° for 24 hours.The nondialyzable material is lyophilyzed and applied to TLC plates asdescribed below.

[1819] Thin-Layer Chromatography: Glycosphingolipids are separated onthin-layer plates of silica gel G, H, or HR. The plates are prepared andactivated at 100° C. for 2-4 hours. Plates of 0.25 mm thickness are usedfor general work and plates of 0.75 mm thickness are used forpreparation of large quantities of material. Thin-layer tanks are linedwith paper and equilibrated with solvent for 4 or more hours before use.Commercial pre-prepared TLC plates (Quantum Industries, Fairfield, N.J.,Brinkman Instruments Inc., Westbury, N.Y., and Analtek Inc., Wilmington,Del.) have been used successfully for qualitative analysis ofglycosphingolipids. Separation on these plates, however, is not usuallyas great as on plates made in the laboratory and contaminants are oftenobtained when silica gel is removed from pre-prepared plates and elutedwith solvents.

[1820] A glycolipid mixture obtained from a column is separated intovarious components on a silica gel H plate (0.25 mm) using achloroform-methanol-water (100:42:6) solvent system. Some hematoside,NANA(2-3)gal(1-4)glc-ceramide, is usually partitioned into the lowerphase of a Folch wash and is separated from human or porcine globoside,galNAc (1-3)gal(1-4)gal(1-4) glc-ceramide in this system. Monohexosylceramide, glc- and gal-ceramide, and dihexosyl ceramide, gal (1→4)glc-ceramide and gal-(1-4) gal-ceramide, often appear as two spotsbecause of the presence of a-hydroxy fatty acids in the ceramide.Otherwise the two forms of monohexosyl and dihexosylceramide are notseparated on silica gel alone. Glucosylceramide and galactosylceramide,however, have been resolved on borate-impregnated thin-layer plates.Sulfatide (galactosylceramide sulfate) is not usually completelyseparated from dihexosylceramide, but these compounds can be completelyseparated by DEAE chromatography. Gangliosides larger than hematosideremain very near the origin in this system.

[1821] Gangliosides and neutral glycosphingolipids with more than fourglycosyl residues are separated by more polar solvent systems such aschloroform-methanol-water (60:45: 10) or chloroform-methanol-2s N NH₄OH(65:45:9). In the latter case, when gangliosides are involved, the plateis developed two times with thorough drying (4 hours at roomtemperature) between developments. Hematoside is well separated fromgloboside on silica gel G plates with this system.

[1822] Glycosphingolipids are visualized with iodine vapor or byspraying with a 2% a-naphthol solution (in ethanol) followed byconcentrated H₂SO₄ spray and heating for 10 minutes at 100°. Thea-naphthol spray gives deep red-purple spots withcarbohydrate-containing compounds and brown spots with phospholipids orneutral lipids. As little as 1-10 mg of material is visualized in thisway. Gangliosides are specifically visualized by spraying with thefollowing solution: mix 10 ml of 3% resorcinol (stored in refrigerator)with 80 ml of concentrated HCl, 0.25 ml of 0.1 M CuSO₄ and enough waterto make 100 ml of solution. The sprayed plate is placed horizontally ina closed jar and heated in an oven at 125° for 20 minutes. Gangliosidesappear as black or purple areas and other compounds appear as lightbrown areas.

[1823] Preparative thin-layer chromatography is carried out by streakingthe sample on a 0.75 mm thick plate and developing as outlined above.Only the edges of the streak are visualized with I₂ or a-naphthol andareas containing neutral glycolipids are removed from the plate and thesilica gel is eluted with chloroform-methanol-water (100:50:10).Gangliosides are eluted from silica gel with more polar solvents such aschloroform-methanol-water (50:50:15).

[1824] DEAE Column Chromatography: Water-soluble oligoglycosylceramidesare separated from gangliosides from fraction II by the followingprocedure. Diethylaminoethyl cellulose (DEAE) in the acetate form iswashed and columns are prepared. The sample is applied inchloroform-methanol (7:3) and neutral glycolipids are eluted with 8 bedvolumes each of chloroform-methanol (7:3) and (1:1). Gangliosides areretained on the column and are eluted with 10 bed volumes ofchloroform-methanol (2:1) saturated with aqueous 58% NH₄OH.

[1825] Dihexosylceramide and sulfatide isolated from a preparative TLCplate as described earlier is separated on a DEAE column. The sample isapplied in chloroform-methanol (9:1) and neutral dihexosylceramide iseluted with 10 bed volumes each of chloroform-methanol (9:1) andchloroform-methanol (7:3). The sulfatide is eluted withchloroform-methanol (4:1) made 10 mM with respect to ammonium acetate,to which is added 20 ml of 28% aqueous ammonia per liter. Sulfateanalysis of lipid fractions is carried out.

[1826] Florisil Column Chromatography: An alternative method ofisolation of glycosphingolipids is by Florisil column chromatography ofthe peracetylated glycolipids wherein all the glycophingolipids (exceptpolysialylgangliosides) are isolated in one fraction. Briefly, theprocedure consists of peracetylation of the total lipid extract withpyridine-acetic anhydride (3:2) (1 ml per 50 mg of dry total lipid). Thepyridine and acetic anhydride are removed in vacuo with additions oftoluene, and the products are applied to a Florisil column (40 g pergram of lipid), and neutral lipids and cholesterol are eluted withdichloroethane (8 bed volumes). Peracetylated glycosphingolipids areeluted with 8 bed volumes of dichloroethane-acetone (1:1), andphospholipids are eluted with 5 bed volumes ofdichloroethane-methanol-water (2:8:1). Acetyl groups are removed fromthe glycolipids with 0.25% sodium methoxide in chloroform methanol (1:1)(1 ml per 25 mg of lipid) at 25° for 30 minutes. The mixture isneutralized with acetic acid, emulsified in water and dialyzed overnightat 40 The glycolipid fraction, analyzed by TLC, is free of contaminatingphospholipids.

[1827] Characterization of Glycosphingolipids: The first step in thecharacterization of glycosphingolipids is the complete cleavage of thelipid into its component parts which is carried out by methanolysis with0.75 N methanolic HCl. The products of methanolysis of aglycosphingolipid ate sphingolipid bases and their O-methyl derivatives,fatty acid methyl esters, and methyl glycosides. These components areseparated by solvent extraction and analyzed by gas-liquidchromatography.

[1828] Methanolysis: A solution of a glycosphingolipid (up to 1 mg),isolated from columns or thin-layer chromatography plates, is evaporatedto dryness in an 8-ml culture tube fitted with a Teflon-lined cap. Threemilliliters of 0.75 N methanolic HCl (prepared by bubbling gaseous HClinto methanol) is added to the sample, and the capped tube is heated at80° for 12-20 hours. At the end of this period, 0.05-0.3 mmole ofmannitol (in methanol) is added as an internal standard. The sample isextracted three times with 1 ml hexane to remove fatty acid methylesters. The hexane solution of methyl esters is retained for GLCanalysis. Approximately 100 mg of solid Ag₂CO₂ is added to each. tubeand carefully mixed until neutral. Methyl glycosides of amino sugars andneuraminyl methyl ester, and sphingosines- are N-acetylated by additionof 1 ml of acetic anhydride. The remaining Ag,C03 and AgG1 act ascatalyst for this reaction. The mixture is allowed to react for 6-16hours at room temperature, after which the sample is centrifuged and theprecipitate is washed with methanol several times. About 0.25 ml of H₂Ois added to decompose excess acetic anhydride, and the sample isevaporated under a stream of nitrogen. If N-acetylation is notperformed, the neutralized sample is centrifuged, washed, and evaporatedto dryness under nitrogen.

[1829] Trimethylsilylation and Gas-Liquid Chromatography of MethylGlycosides: Dry samples of methyl glycosides are converted totrimethylsilyl (TMS) derivatives by addition ofpyridine-hexamethyldisilazane-tri-methylchlorosilane (8:2:1) (about 50mM for 500 mg of lipid). The mixture is allowed to stand for 15 minutesat room temperature and an aliquot is injected into the gas-liquidchromatograph. The mixed silane solution is cloudy and is used withoutcentrifugation, but exposure to water vapor is avoided. If very smallamounts of sugars are present, the sample is evaporated under nitrogenand redissolved in a convenient solvent such as pyridine or CS₂.

[1830] An aliquot of the solution of TMS derivatives is injected into agas-liquid chromatographic column (2 m by 3 mm) of 3% SE-30 or OV-1 onSupelcoport (80/100 mesh, Supelco Inc., Bellefonte, Pa.) at 160° withnitrogen carrier gas (25 ml/minute). Programming from 1500 to 2500 at 30per minute is useful when sialic acids are present. A chromatogram of amethanolyzed sample of globoside, galNAc(1→3)gal (1→4) gal (1→4)glc-ceramide shows three peaks for TMS methyl-D-galactoside (a, g, and bforms); two peaks for methyl-D-glucoside (a and b forms); and two majorpeaks for methyl-2-acetamido-2-deoxy-D-galactoside. Other methods areuseful for gas-liquid chromatography of methyl glycosides are suitablefor identification of glycolipids containing fucose, glucose, galactose,galactosamine, glucosamine, and sialic acid. Mannose exhibits peaksoverlapping with galactose and if these two sugars are present, themethod employing alditol acetates is preferred.

[1831] The ratios of glucose and galactose are determined withoutconversion factors by simply comparing the ratio of the total peak areasof each methyl glycoside. Since many glycosphingolipids contain only oneglucose, ratios are usually expressed in relation to glucose and forgloboside the ratio of galactose to glucose is 2. The ratio ofgalactosamine to glucose calculated in this way is usually about 0.65for globoside. Methanolysis, N-acetylation, and trimethylsilylation iscarried out to obtain reproducible ratios for hexosamines. The massratio obtained for N-acetylneuraminic acid to glucose is usually 1.0 to1.2, and these values should be compared with those obtained from knowngangliosides treated in the same way. The absolute quantity of galactoseand glucose are determined by comparison to the internal standardmannitol with the use of the following equation.

mmoles glucose=area of glucose peaks/area of mannitol peak×1.25×mmolesof mannitol added

[1832] The mannitol peak falls between the second glucose peak and thefirst galactosamine peak and does not interfere with either compound.The area of peaks is calculated by triangulation.

[1833] Fatty Acids and Sphingosines: Normal fatty acids and a-hydroxyfatty acids are determined qualitatively and quantitatively bygas-liquid chromatography of the fatty acid methyl esters obtained fromthe hexane extract of the methanolyzate. Sphingosines are determined byhydrolysis of the glycolipid with aqueous HCl followed by N-acetylationand GLC of the TMS derivatives. A colorimetric assay and a methodinvolving GLC of aldehydes produced by NaIO4 cleavage of sphingosine arealso available.

[1834] Enzymatic Degradation of Glycosphingolipids: Specificglycosidases are used for sequence determination and anomeric analysisof glycolipids. Glycosyl residues are released sequentially fromgloboside (cytolipin R reacts in the same way) by stepwise treatmentwith the following glycosidases; β-hexosaminidase from jack bean, a-galactosidase from fig ficin, and β-galactosidase from jack bean. Reactionsare carried out with 100 mg of lipid in 0.1 ml of 0.1 M sodium citratebuffer at pH 5, containing 100 mg of crude ox bile sodium taurocholate.After 18 hours at 37°, reaction mixtures are frozen and lyophilized. Onemilliliter of chloroform-methanol. (2:1) is added and the mixture issonicated for 5 minutes. After centrifugation, the supernatant fractionis dried, taken up in a small amount of chloroform-methanol (2:1) andspotted on a silica gel HR plate. The plate is developed inchloroform-methanopwater (100:42:6) and visualized with 12 vapors ora-naphthol spray. Products are identified by cochromatography withstandards and by elution, methanolysis and GLC analysis.

[1835] Mass Spectrometry of TMS Glycosphingolipids: Mass spectrometry ofintact TMS derivatives of glycolipids gives information about the sugargroups, the fatty acid and the sphingosine portion ofglycosphingolipids. Bis(trimethylsilyl)trifluoroacetamide (100 ml) andpyridine (50 ml) are added to 20-200 mg of the purifiedglycosphingolipid in a small capped vial and heated at 60° for about 30minutes. An aliquot containing 10-20 mg of the TMS glycolipid isevaporated to dryness under nitrogen in a mass spectrometer direct probetube. The samples are volatilized in the mass spectrometer ion source attemperatures ranging from 10° to 180° depending on the size of theoligosaccharide unit. The following information can be obtained bycomparison of the resulting mass spectra with those of referencesamples: (1) whether the terminal residue is a hexose or hexosamine; (2)the number of and nature of —N-acetylneuraminic acid groups (i.e.,terminal or branched); (3) whether N-acetyl and/or N-glycolylneuraminateis present; (4) information regarding the number of glycosyl residuespresent and the fatty acid and sphingosine composition. Because of thethe inability to distinguish between hexoses, this technique inconjunction with other techniques, such as permethylation analyses, andstudies with specific glycosidases.

[1836] Ozonolysis of Glycosphingolipids: The carbohydrate portion ofglycosphingolipids is cleaved from the lipid portion. Theglucose-sphingosine linkage is broken but there is no hydrolysis ofother glycosidic linkages, including those of sialic acid residues. Theglycolipid (100 mg) is ozonized in 50 ml of methanol at roomtemperature. Ozone consumption is monitored by bubbling the effluent gasthrough a KI-starch solution which blackens when excess ozone ispresent. The solution is dried in vacuo and the compound is hydrolyzedwith 10 ml of 0.2 M Na₂CO₂, for 12 hours at 20°. Sodium ion is removedby stirring with Dowex 50 (H+) and the resin is filtered. After a Foichpartition, the upper aqueous phase is lyophilized and the resultantoligosaccharides (about 80% yield) are stored in a desiccator. Theprocedure is changed for microscale operation (1 mg of lipid).

[1837] Permethylation of Glycosphingolipids: Permethylation, hydrolysis,and gas-liquid chromatography of glycosphingolipids is used to determinelinkage of glycosyl residues. Permethylation is carried out using methylsulfinyl carbanion. Sodium hydride (0.88 g of 57% in oil) is washed sixtimes under nitrogen with dry hexane, drained thoroughly, and stirredwith dimethyl sulfoxide (10 ml) under a stream of nitrogen at 70° for 3hours or until bubbling ceases and the solution turns a dark cleargreen. Any dark precipitate is removed at this point by centrifugation.The carbanion solution (about 0.5 ml) is added under a stream ofnitrogen to the glycolipid sample in 0.5 ml of dimethyl sulfoxide, andthe mixture is sonicated briefly. After standing at room temperature for2-6 hours, 1.5 ml of CH3I is added dropwise under nitrogen, and themixture is allowed to react for 1 hour. After this step, it is notnecessary to keep the reaction dry. The permethylated glycolipids areextracted into chloroform, the chloroform layer is washed once with 1%Na2S3O3 to remove I₂, and four times with water. The chloroform fractionis mixed with absolute ethanol and is evaporated under nitrogen. Twomilliliters of 1 N H2S04 is added, and after heating at 105° for 12hours the hydrolyzate is neutralized with BaCO₃ diluted, and filtered onCelite and filter paper, washing the Celite twice with water (5 ml). Thesample is concentrated to 5 ml and percolated onto a small Dowex 50 H+column. Neutral sugars are eluted with water and methanol-water (1:3)(10 ml each) and amino sugars are eluted with 0.3 N NH4OH. The resultingpartially methylated sugars are reduced with NaBH4, the products areacetylated and gas-liquid chromatography is carried out.

[1838] Partial Hydrolysis of Glycosphingolipids: The presence ofN-acetylneuraminic acid or N-glycolylneuraminic acid in a gangliosidesample is determined by mild acid hydrolysis of the neuraminic acidfollowed by TLC or GLO analysis. The N-acylneuraminic acids are releasedfrom gangliosides (0.1-0.2 mg) with 1 ml of 0.05 M HCl (aqueous HCl) at80° for 1 hour. The solution is extracted with chloroform and theaqueous phase is percolated through about 2 g of Dowex 1 (acetate form)in a small column. The column is washed with 8 ml of water, and theneuraminic acids are eluted with 10 ml of 1 M formic acid. The sample isanalyzed on silica gel C plates using n-propanol-water-concentratedammonia (6:2:1). The Rf of N-acetylneuraminic acid is 0.41 and that ofN-glycolylneuraminic acid is 0.28. This sample is also be derivatizedwith bis (trimethylsilyl) trifloroacetamide and analyzed by GLC.

[1839] Neuraminic acids are usually analyzed by GLC as their TMS-methylketoside methyl ester derivatives. A ganglioside sample (0.1 mg) ormixture of glycolipids is methanolyzed with 2 ml of 0.05 M methanolicHCl (1 part 12 N HCl and 240 parts methanol) at 80° for 1 hour. Thecooled solution is extracted three times with 3 ml hexane and themethanolic layer is evaporated to dryness under nitrogen.Pyridine-hexamethyldisilazane-trimethylchlorosilane (8:4:2) (50 ml) isadded and the TMS derivatives are analyzed on a 3% OV-1 column (2 m×2mm) at 205°. The sulfate moiety of sulfatide (up to 2 mg) is released byreaction with 2 ml of 0.05 N methanolic HCl for 4 hours at roomtemperature. The reaction mixture is neutralized with aqueous NaOH andthe glycolipid product is extracted by Folch partition with 4 ml ofchloroform. The chloroform layer is washed with 2 ml of methanol-water(1:1) and is analyzed by TLC.

[1840] The carbohydrate sequence of glycosphingolipids is determined bypartial degradation under mild acidic conditions. The glycolipid (1 mg)is hydrolyzed with 1 ml of 0.3 N HCl in chloroform-methanol (2:1) at 60°for various times up to 2 hours. After Folch partition (with addition of0.2 volume of water) the lower phase is dried in vacuo and the upperphase is deionized with Amberlite CG-4B resin (OH form). The glycolipids(from lower phase) are purified by TLC and analyzed by GLC aftercomplete hydrolysis. The upper phase is analyzed for sugars (andpolysaccharides) by GLC. Trihexosyl ceramide, gal(1→4)gal (1→4)glcceramide, hydrolyzed for 2 hours in this way did not yield ceramide butyielded cerebroside containing only glucose, and a dihexosyl ceramidecontaining glucose and galactose in a 1:1 molar ratio. The water-solublefraction contained galactose and a disaccharide but no glucose. Thesedata provide evidence for a linear arrangement of the hexose units asgiven above.

[1841] Characterization of the oligosaccharide moieties of glycolipidsby partial degradation is also been carried out by treatment ofglycolipids with 0.1 N aqueous HCl for 30 minutes to 3 hours, and bytreatment of gangliosides with 0.01 N H₂SO₄ at 85° for 1 hour and 0.1 NH₂SO₄ at 100° for 1 hour.”

[1842] Mixed Molecular Species of Glycosphingolipids: The purificationand characterization of glycosphingolipids is complicated by the factthat naturally occurring glycosphingolipids which are homogeneous in thecarbohydrate portion are heterogeneous in the sphingosine and fatty acidportions. Complete hydrolysis and subsequent analyses of the fatty acidsand long-chain bases provide information about the extent of thisheterogeneity. Additional heterogeneity results when there areglycosphingolipids with identical composition but which differ inglycosidic linkage and/or anomeric configuration of one or moreglycosidic bonds. Such mixtures probably cannot be separated by TLCalone. Whether or not products which are homogeneous by TLC actuallycontain such mixtures can only be determined with techniques such aspermethylation and sequential enzymatic degradation.

[1843] Extraction of Inositolphosphorylceramides (InsPCers) of Fungi andYeast: Extraction of InsPCers from Saccharomyces cerevisiae and from themycelial phase of Neurospora crassa is as follows: Cells uniformlylabeled with [³H]inositol are used to gauge the extraction efficiency ofa variety of procedures. Treat the cells with 5% trichloroacetic acid at0C to destroy phospholipases, known to be activated by organic solventsduring lipid extraction, followed by extraction with a slightlyalkaline, warm, water-rich mixture ofethanol-diethylether-water-pyridine. This method approached 100%extraction of the labeled inositol; Some water-poor solvents completelyfailed to extract the InsPCers. The adopted procedure has been used forInsPCer extraction from other fungi such as Histoplasma capsulatum andfrom fresh tobacco leaves and has been used for the extraction oflipophosphoglycan from Leishmania donovani. In the absence of labelingof InsPCer components, efficacy of InsPCer extraction could be judged bymonitoring total long-chain base and/or very-long-chain fatty acids.

[1844] Purification of InsPCers: Purification of INsPCers is nodifferent from any very polar, acidic lipid. First, InsPCers precipitatealmost quantitatively at low temperature after adjusting to pH 5 theinitial lipid extracts from S. cerevisiae, H. capsulatum, N. crassa, andtobacco leaves. Second, mild alkaline methanolysis is used to destroythe ester-containing lipids in crude or semipurified extracts; however,stronger alkaline conditions should be avoided owing to the lability ofunsubstituted inositol attached by a phosphodiester bond. Finally,liquid chromatography on silica gel columns is used for isolatingInsPCers; low levels of salt in the eluting solvents are required tochromatograph macroscopic quantities of the very polar InsPCers. Asolvent polar enough to dissolve a practical amount of the very polarInsPCers yields insufficient retention. Inclusion of salt increases theretention presumably by providing a charged surface for the negativelycharged InsPCers. In contrast, the salt had little influence on theretention of neutral lipids.

[1845] Extraction and Purification of Galactosylceramides--Cell culture:Human kidney cells (PT cells) are prepared as described previously(Chatteree et al., Proc. Natl. Acad. Sci. (1983)). Human cadaver kidneys(post mortem <12 hr) are used for the isolation of PT cells.Glucosylceramide was prepared from the spleen of a Gaucher's patient.Digalactosyklceramide is prepared from the kidney of a Fabry's patient.Other GSLs are purchased from Sigma. Normal rat kidney cells werepurchased from the American Tissue Culture Collection (Rockville, Md.).Cells (×10⁴) are seeded in 24 well trays and grown for 6 days in mediumcontaining 10% fetal calf serutn (Hyclone, Utah).

[1846] Lipid Extraction and Fractionation of GSL from Human Kidney:Total lipids are extracted from freeze-dried cultured PT cells or humankidney cortex or both by vigorous homogenization and extraction with hot(50° C.) chloroform:methanol 2:1 (v/v)10 ml/mg protein. The lipidextracts are filtered on a sintered disc tunnel and the non-lipidresidue is extracted further with hot chloroform-methanol 1:2 (v/v)containing 5% H20. Lipid extraction is pursued at 50° C. for 30 min withconstant stirring. The lipid extract is filtered on a sintered glassfunnel and the residue is extracted further, first with hotchloroform:methanol:water 1:2, 5%) and then with methanol. The lipidextracts are pooled and dried by flash evaporation. Water-solublecontaminants are removed from the lipid extracts.

[1847] Isolation and Purification of GSL: The lower phase lipid fractionobtained following Folch partitioning of human kidney total lipids isfractionated into neutral lipid, GSL and phospholipids by silicic acidchromatography as described previously (Chatterjee et al., 1982). Theacetone:methanol fraction containing GSL is subjected to alkalinemethanolysis, neutralized and dried under a nitrogen atmosphere. Thedried residues are solubilized in chloroform:methanol 2:1 (vlv) andsubjected to HPTLC on Silica gel HPTLC plates withchloroform:methanol:water (65:25:4, v/v) as the developing solvent. Theindividual GSLs are identified with aniline diphenylamine (OPA) reagentor iodine vapours. The chromatogram is calibrated with authentic GSLstandards of known structure. The total GSL fraction was precipitatedwith ether and subjected to mild alkali-catalysed methanolysis, dialyzedagainst water and separated by silicic acid chromatography. The silicicacid column is equilibrated with chloroform:methanol (19:1) and a samplesuspended in the same solvent is applied on the column bed. The columnis eluted successively with 12, 14, 16, 20, 30 and 50% methanol inchloroform, and the fractions are dried in a nitrogen atmosphere. Thevarious fractions are analysed for the composition of GSLs. Several ofthe fractions obtained when the column is eluted with 14% methanol inchloroform contained digalactosylceramide. Such preparations ofdigalactosylceramide are further characterized by high-performanceliquid chromatography (HPLC) and are utilized for binding to SEB.

[1848] Quantitation of GSL: GSLs are quantified by HPLC, afterperbenzoylation. An aliquot of perbenzoylated GSL sample is suspended inhexane and subjected to HPLC on a Spherisorb Si-5 column with detectionat 230 nm. The amount of GSL is calculated by using a standard curve forthe respective GSLs.

[1849] Characterization of Galactosylceramides: Acid hydrolysis of GSLsare carried out followed by TLC of sugars on aluminium-backed silica gel60 (without indicator) HPTLC plates with the use of 2-propanol-1% sodiumborate (3:1, v/v) as the developing solvent. The sugars are localized byspraying the plate with 10% H2S04 in 50% ethanol and heating at 150° C.for 10 min in an incubator. Anomeric linkage of the purified GSLreceptor is determined using β-galactosidase, α-galactosidase andβ-glucosidase An aliquot of the purified GSL receptor is incubated withor without β-galactosidase; β-galactosidase and β-glucosidase in 0.05 Mcitrate buffer (pH 5.4) containing taurodeoxycholate for 18 h at 37° C.The reaction is terminated with chloroform:methanol (2:1 vlv) and thelower-chloroform layer is subjected to HPTLC. The plate is developedwith aniline DPA reagent.

[1850] GC-MS of GSL: Suitable aliquots of the putative SEB receptor aresubjected to acid-catalysed methanolysis (Esselman et at., 1972). Themethyl sugars, methyl fatty acids and methyl sphingosines are purifiedby solvent extraction as described above. They were dried in a nitrogenatmosphere and derivatized by employing trimethylchlorosilane reagent.The derivatized samples (fatty acid methyl esters) suspended in hexanewere injected into a Varian-3400 gas chromatograph (DB-wax capillarycolumn, 30 m; J and W Company, California) that is attached to a massspectrometer ITD-850, Finnigan Ion Trap detector. Helium is used as acarrier gas. Temperature programming from 160° C. to 250° C. at 1°C./min is employed to separate the various fatty acid methyl esters. TheTMSi sugars are separated on a DB-5 column using temperature programmingfrom 160° C. to 250° C. at 1° C./min. Data analysis of TMSi sugars ispursued by the use of a Compaq deskpro-2862 computer. Suitable aliquotsof the GSL are subjected to microscale permethylation. The gaschromatography column (DB-5 capillary column 0.25 mm×30 m) is calibratedwith authentic standards of mixtures of partially methylated alditolacetates (Biocarb. Chemicals, Sweden). Temperature programming is from160° C. to 250° C. at 1° C./min and from 250° C. to 350° C. at 2°C./min.

EXAMPLE 56 Transfection of Thymidine Kinase Gene into ActivatedImmunocytes

[1851] In order that the immunocytes with deleted or inactivated IRs donot undergo unlimited proliferation in vivo it is necessary to provide amethod for eliminating these cells after they have performed theirtumoricidal function in vivo. To achieve this effect, aretrovirus-mediated transfer of a gene encoding a ‘prodrug’, a reagentthat confers sensitivity to cell killing following subsequentadministration of a suitable drug is used. Thymidine kinase (Wigler M etal., Cell, 11: 223 (1977); Colbere-Garapin F et al., Proc. Natl. Acad.Sci., 76: 3755 (1979)) is encoded by the cellular, HSV, or vacciniavirus tk genes and the HSV-tk gene is used as a prodrug gene. The HSV-tkgene is transfected into immunocytes by methods given in Example 1. TheHSV-tk confers sensitivity to the drug gancyclovir by phosphorylating itwithin the cell to form gancyclovir monophosphate which is subsequentlyconverted by cellular kinases to gancyclovir triphosphate. This compoundinhibits DNA polymerase and causes cell death. The immunocytes areadministered to the host. Unopposed proliferation of immunocytes cellsdeleted of IR_(TAA)s, IRS_(SAg)s, IR_(IDA)s in response to tumorassociated lipid-based antigens may lead to immunocyte excess.Therefore, after the immunocytes have performed their tumor killing invivo, gancyclovir is administered in conventional pharmacologic doseswhich induces apoptosis of HSV-tk transfected immunocytes.

EXAMPLE 57 Deletion of Immunocyte and Antigen Presenting Cell LipTAAReceptors in Tumor Bearing Hosts

[1852] Lip-TAA receptors are deleted or inactivated in T Cells, NKTCells and NK Cells in vivo and in vitro using but not limited toanti-sense oligonucleotides gene knockouts Examples 51-52. Immunocytesand accessory cells used for adoptive transfer are depleted of their TGGreceptors using antisense and gene knockout technology as given inExamples 51-52. The T Cells, NKT Cells and NK Cells (collectivelyimmunocytes) and accessory or antigen presenting cells including but notlimited to macrophages, dendritic cells, fibroblasts, B cells and withdeleted or inactivated TGG receptors are therefore protected fromganglioside-induced immune suppression and are capable of inducing ananti-tumor response.

[1853] Model Tumor Systems for Testing Efficacy of Immunocytes Deletedof Lip-TAA: Receptors: Tumor models used for determining efficacy ofganglioside receptor deletion or inhibition on immunocytes and accessorycells are given in Examples 20-23.

[1854] The deletion or inhibition of ganglioside receptors onimmunocytes or accessory cells in tumor bearing hosts is carried out invivo. Alternatively, the deletion or inhibition of ganglioside receptorson immunocytes or accessory cells can be achieved ex vivo and thetreated cells used for adoptive immunotherapy. Outcomes are given inExamples 21, 23.

[1855] General Procedures for Ex Vivo Sensitization of ImmunocytesDepleted of Lip-TAA Receptors to Produce Tumor Specific Effector Cellsfor Adoptive Immunotherapy

[1856] MCA 205/207 tumor cells, A/20 lymphoma cells, FBL-3, a Friendvirus-induced erythroleukemia of B6 origin, is (provided by Dr. PhilipGreenberg) and Lewis lung carcinoma cells are used. Tumor growth isinitiated by subcutaneous inoculation of mice on both flanks with1.5×10⁶ tumor cells suspended in 0.05 ml of HBSS. After 9-12 days oftumor growth (approximately 8 mm in diameter), tumor-draining inguinalLN are removed sterilely. Lymphocyte suspensions are prepared by teasingLN with needles followed by pressing with the blunt end of a 10-miplastic syringe in HBSS. Tumor draining LN cells delpleted ofganglioside receptors are stimulated in vitro in a two-step procedure.Briefly, 4×10⁶ LN cells in 2ml of complete medium (CM) containing thesuperantigens are incubated in a well of 24-well plates at 37° C. in a5% C0₂ atmosphere for 2 days. CM consisted of RPMI 1640 mediumsupplemented with 10% heat-inactivated FCS, 0.1 mM nonessential aminoacids, 1 mM sodium pyruvate, 2 mM freshly prepared L-glutamine. 100ug/ml streptomycin, 100 U/ml penicillin, a 50 mg/ml gentamycin, 0.55mg/ml fungizone (all from GIBCO, GrandIsland, N.Y.) and 5×10⁻⁵ M2-mercaptoethanol (Sigma). The cells are harvested, then washed andfurther cultured a 3×10⁵/well in 2 ml of CM with IL-2. After 3-dayincubation in IL-2, the cells are collected and counted to determine thedegree of proliferation. Finally, the cells are suspended in appropriatemedia for flow cytometric analysis, evaluation of cytotoxicity andlymphokine secretion, or for adoptive immunotherapy.

[1857] General Adoptive Immunotherapy Protocol: Mice are injected with 2to 3×10⁵ syngeneic MCA 205 or 207 fibrosarcaoma tumor cells suspended in1 ml of HBSS to initiate pulmonary metastases. This is the preferredanimal model for evaluation of the adoptively transferred effector cells(Shu S. a at, .I. Immunol. 152: 1277-1288 (1994)). Other models of tumormetastases are induced by injection of:

[1858] (a) with 2×10⁶ B16 or FBL-3 (erythroleukemia) cells in the spleento induce liver metastasis,

[1859] (b) intravenously with 3×10⁵ B16 or 2×10⁶ LLC (Lewis lungcarcinoma) cells for pulmonary metastases or;

[1860] (c) subcutaneously with 2×10⁶ B 16 cells (melanoma) forsubcutaneous tumor growth on day 0.

[1861] On day 3, ganglioside receptor depleted immunocytes or accessorycells are given i.v. at numbers indicated generally 10⁶-10⁸ intosyngeneic hosts. In some instances, mice are also treated with 15,000 UIL2 in 0.5 ml HBSS twice daily for 4 consecutive days to promote the invivo function and survival of the activated cells. On day 20 or 21, allmice are randomized, killed and metastatic tumor nodules enumerated. Ifpulmonary metastases exceeds 250, this number is arbitrarily assignedfor statistical analysis. The significance of differences in metastasesnumbers between experimental group is determined by the Wilcoxon ranksum test. Two sided p values of <0.1 are considered significant. Eachexperimental group consists of at least five mice and no animal isexcluded from the statistical evaluation.

EXAMPLE 58

[1862] The Lip-TAAs expressed and shed by tumor cells are isolated andtested for capacity to bind to various lipid binding proteins and toinhibit mitogen induced T cell proliferation. The best binding agent foruse in vivo as a decoy to bind Lip-TAAs and prevent their uptake by APCsof T cell is selected from among the Siglecs, prosaposins, saposins,glycolipid transfer proteins (GLTP), non-specific lipid transfer agentsand other ganglioside binding proteins. Monoclonal antibodies specificfor Lip-TAAs are also useful as Lip-TAA binding agents. They areparticularly useful when it is determined that immunosuppressiveLip-TAAs share a common antigenic domain. The natural Lip-TAA bindingagents are preferred because of their broad specificity and unlikemonoclonal antibodies do not require preparation of a new agent for eachvariant immunosuppressive ganglioside identified.

[1863] Extraction of gangliosides from tumors.: Gangliosides arepurified from pools of human tumors and/or serum lipoprotein fractionsas previously described. Briefly, tumor tissue is homogenized inchloroform/methanol 1/1 (v/v) and extracted twice in the same solvent.After evaporation the lipid residue is dissolved in chloroform/methanol2/1, filtered and dried to remove most peptidic contaminants. After twocycles, total gangliosides are purified by three successive partitionsin chloroform/methanol/phosphate-buffered saline pH 7.40, 1/1/0.7(v/v/v) and desalting by reverse-phase chromatography on C 18-bondedsilica gel. In the case of serum or serum fractions, the procedure isessentially the same but contained only one extraction. Purity of thepreparation was controlled by thin-layer chromatography (TLC) on silicagel 60 HPTLC plates (Merck. Paris. France) using a solvent system madeof chloroform/methanol/0.2% aqueous CaCl₂. 60/35/8 (v/v/v). Staining isperformed with resorcinol HCl. Lipid-bound sialic acid is assayed by thepenodate/resorcinol method. The relative proportion of each gangliosideis estimated after resorcinol/HCl by scanning densitometry of the TLCplates on a Shimadzu CS-930 Chromatoscan set at 580 nm.

[1864] Preparation of lipoproteiin fractions: Lipoprotein fractions areprepared from human AB sera by sequential centrifugations. Thelipoprotein is fractionated by density-gradient ultracentrifugation insodium bromide. Sera are first centrifuged at 10,000 g for 20 minutes toremove chylomicrons. Very-low-density lipoprotein (VLDL), low densitylipoprotein (LDL) and high-density lipoprotein (HDL) are floated atrespective densities of 1.006. 1.073 and 1.220 g/ml. LDL HDL and thelipoprotein-free fraction (LFF) are extensively dialyzed againstphosphate buffer before use to remove sodium bromide and resuspended inphosphate buffered saline in the original volume of serum from whichthey are extracted. Total serum and the serum lipoprotein fractions areat a final concentration of 10% (v/v).

[1865] Inhibition of peripheral blood mononuclear cell (PBMC)proliferation: PBMC are obtained from the blood of normal donors byFicoll/Hypaque centrifugation and are washed in medium without serum(RPMI-1640. Gibco, with 1 mM glutamine and penicillin/streptomycin). Theculture medium consists of medium without serum, or medium supplemented(10% v/v) with one of the following: total human AB serum, VLDL, LDL,HDL or LFF. Samples containing 1×10⁵ cells are incubated in a finalvolume of 200 μl (96-well plates, Beckton-Dickinson. Le Pont de Claix.France) in the presence or absence of total tumor gangliosides and oneof the following mitogens: phytohemagglutinin (PHA, 1 μg/m; Sigma. StLouis, Mo.), OKT3 monoclonal antibody (100 ng/ml; Coulter) concanavalinA (0.5 μg/ml; Sigma), interleukin-2 (IL-2. 1000 UI/ml, Roche, France) or12-O-tetradecanoylphorbol 13-acetate (10 ng/ml: Sigma). After 72 h ofincubation, proliferation is assayed by the incorporation of[³H]thymidine (0.5 mCi/well, specific activity 1 mCi/nmol=37 GBq/mmol;C.E.A., Gif-sur-Yvette, France) after an 18-h pulse.

[1866] Screening of Lip-TAAs for Binding to Lip-TAA Binding Proteins:The extracellular domains of Siglecs or other LipTAA binding agents,e.g., glycolipid transfer proteins (GLTP), prosaposins are cloned into aFLAG-human Fc expression vector (pEDdc). COS-7 cells are transientlytransfected at 60-70% confluency using LipofectAMINE Reagent (LifeTechnologies), in serum-free OptiMEM medium. After 5 h the medium isdiluted 2 times with 10% fetal calf serum containing OptiMEM medium andthe next day the medium is changed for OptiMEM with 2% fetal calf serum.The COS-7 cell supernatants are collected 5-7 days after transfection.The fusion protein is purified on Protein A-Sepharose. Microtiter wells(Nunc) are coated overnight at 4° C. with Protein A (200 ng/well) in 50mM carbonate/bicarbonate buffer, pH 9.5. Wells are blocked withenzyme-linked immunosorbent assay buffer (20 mM HEPES, 1% bovine serumalbumin, 125 mM NaCl, 1 mM EDTA, pH 7.45) for 1 h and incubated withvarious lipid binding agents (500 ng/well) for 2 h. Between incubations(all at room temperature) wells are washed 3 times with enzyme-linkedimmunosorbent assay buffer. Lip-TAAs and circulating lipoprotein-boundTAAs are isolated and frationated from tumors and tumor bearing sera asdescribed in Example 59 and below in this section. They are are addedfor 2 h at various concentrations ranging from 100 ng to 2 μg/well. Theplates are washed and biotin-conjugated polyacrylamide substituted withthe natural substrates for the individual lipid binding agent are added.In the case of Siglec 5 this is Neu5Acα2-6Gal/β-4G1c,Neu5Acα2-3Galβ1-4G1c, Neu5Acα2-6GalNAcα(sialyl-Tn), and GalNAcα(Tn)(Glycotech). The plates are washed then incubated with alkalinephosphatase-conjugated streptavidin (Life Technologies; 1:1000) for 1 hand development with 100 pl/well of p-nitrophenyl phosphate LiquidSubstrate System (Sigma). Plates are read-out at 405 nm. Competitiveinhibition of binding of the Lip-TAA relative to the natural substatefor each lipid binding agent is recorded. Concomitantly, the Lip-TAAsand circulating lipoprotein fractions are tested for their capacity toinhibit mitogen induced T cell proliferation using the methods givenbelow.

[1867] Preparation of Siglecs from cDNAs: Siglecs 1-7 are isolated bystandard recombinant techniques using cDNA for each siglec. cDNAs forthe Siglecs are obtained starting with their full length amino acidsequences as given in GenBank. Accession numbers for each Siglec isgiven below. The first 120 amino acid sequences of mouse Siglec-1(GenBank accession #Z36293) and human Siglecs 266 (X59350, 4502654,M29273, 4502658, and 4502656, respectively) are used to carry outhomology searches of the dbEST division of GenBank database.

[1868] cDNA of Myelin Associated Glycoprotein, Sialoadhesin, CD22 &CD33: Myclin associated glycoprotein (MAG, Siglec 4a) cDNA has beencloned as described by by Arquint M et al., Proc. Natl. Acad. Sci. 84:600-604 (1987). Sialoadhesin (Siglec 1) cDNA has been cloned by methodsgiven in Crocker P R et al., EMBO J 13: 4490-4503 (1994). The cDNA forthe sialoadhesin p75/ARM1 has been cloned (Falco M et al., J. Exp. Med.190: 793-801 (1999). The cloning of cDNA of CD22 uses methodolgy ofStamenlovic I et al., Nature 345: 74-77 (1990) and Wilson G L et al., J.Exp. Med. 173:137-146 (1991).

[1869] CD33 has been cloned (Cornish A L et al., Blood 92: 2123-2132(1998). cDNA cloning in the human and mouse shows that CD33 is a type Itransmembrane glycoprotein with two Ig-like extracellular domains. Thesedomains belong to the V-set and C2-set of Ig-like domains and sharesequence similarity with a distinctive subgroup of the Ig superfamily.This consists of the myelin-associated glycoprotein (MAG), the Schwanncell myelin protein, the B-cell antigen CD22, and the macrophagereceptor sialoadhesin. All of these Ig-related proteins contain anunusual arrangement of conserved cysteine residues at the NH2 terminus.These are predicted to give rise to an intrasheet disulfide bridgewithin the V-like domain and an interdomain disulfide bridge between theV- and C2-like domains. In addition, MAG, CD33, and CD22 are closelylinked within the genome, mapping within the region of chromosome19q13.1-13.3 in the human and a syntenic region on chromosome 7 of themouse

[1870] cDNA of Siglec-5: By using the amino acid sequence of CD33, aspecific homology search was performed against a database containingmore than one million expression sequence tags (ESTs) obtained from over700 different cDNA libraries. A full-length clone in pBluescript II (SK1encoding one of the CD33-like sequences identified in the search wasisolated from a human activated monocyte library and designated pHMQCD14. A computer search of nucleotide and protein sequence was performedby using the Blast GeneSearch (National Center for BiotechnologyInformation, National Institutes of Health. Bethesda, Md.). Manipulationof sequences and alignments were performed by using Baylor College ofMedicine molecular biology software available on the internet (HumanGenome Center, Baylor College of Medicine, Houston, Tex.).

[1871] Preparation of recombinant Siglec-5: The extracellular region ofsiglec-5 is amplified by polymerase chain reaction (PCR) with thefollowing forward and reverse primers (5′-3′):ACTCTAGAGTTCGATCTCCCTTGCAGCAG and ACAGATCTGTTCGATCTCCCTTGCAGCAG. The PCRproduct is cloned in-frame into the pIGplus vector, which encodes aFactor Xa cleavage site between the extracellular region and the hingeregion of the Fc portion of human IgGI. Plasmids encoding otherFc-proteins are prepared. To generate recombinant proteins, plasmids aretransfected into COS-1 cells by diethylaminoethyl-dextran (DEAE)transfection and Fc-proteins are purified from the conditionedsupernatants. Briefly, supernatant is passed over a protein-A Sepharosecolumn, and the bound protein is eluted with 0.1 mol/L glycine pH 3.0followed by neutralization with 10% vol/vol 1.0 moL/L Tris pH 8.0. Theproteins are dialyzed against 20 mmol/L Tris pH 8.0, and theconcentrations are estimated by using the bicinchoninic acid (BCA) assaykit (Pierce, Rockford, Ill.) with bovine semm albumin (BSA) as astandard. Fc-proteins are shown to be greater than 95% pure. by sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis.

[1872] cDNA of Siglec 6 (OB-BP1 and OB-BP2): A TF-1 cDNA library isconstructed with oligo(dT) priming and cloned into the EcoRI/NotI sitesof the retroviral vector pBabe-X-puro, in which the multiple cloningsite from pBabe-X are substituted for that of pBabe-puro. The library ispackaged in B05C23 cells and then introduced into Ba/F3 cells byretroviral infection. For expression cloning, three rounds of sortingare necessary to isolate a homogenous population positive for rhOB-Fstaining. Geonmic DNA is extracted from the third sort of the Ba/F3cells using the Easy DNA kit (Invitrogen). Polymerase chain reaction isperformed with Vent polymerase (New England Biolabs) using nestedprimers designed from the retroviral vector. Another related moleculewas cloned from the TF-1 library in pBabe-X-puro by colony hybridizationusing the OB-BP1 cDNA as a probe. After the initial submission of thesesequences to GenBank other reports indicated that OB-BP1 (GenBankaccession number U71382) is identical to GDSSL (GenBank accession numberD86358) and OB-BP2 (GenBank accession number U71383) is identical toSiglec-5

[1873] cDNA of Siglec-7: Since the clone lacked a coding sequence forthe first half of an Ig-like domain, 5′-RACE and RT-PCR are performed,using human PBMC total mRNA as a template (PBMC are chosen based onpreliminary Northern analysis using the partial clone). RT-PCR isperformed using primers based on the DNA sequence of the EST clone and5′ RACE product. First strand cDNA is synthesized by reversetranscription of I μg of PBMC total RNA with random DNA hexamers asprimer. The reaction product is subjected to two rounds of PCR using twoDNA polymerase (Roche), using SLX-5′UTR(5′-CTCG-GATCCCTGGCACCTCTAACCC-3′) and SLX-3′UTR(5′-GGTCTAGAACCCTCAAACAAGCCC-3′) as primers. The PCR products aredigested with BamHI and XbaI ligated to BamHI-XbaI sites of pBluescriptII KS(−) (Stratagene) and sequenced. A point mutation converting an Argresidue (R124) to Lys is introduced using QuickChange Site-DirectedMutagenesis Kit (Stratagene), following the manufacturer's protocol. Ahuman 12-lane Multiple Tissue Northern Blot is probed with the NcoI-XhoIfragment of the EST clone labeled with the Strip-EZ DNA kit (Ambion) and[α-³²]dATP (NEN). Hybridization signals are visualized using a PhosphorImager.

[1874] The full-length clone contains an open reading frame encoding 374amino acids, starting with a typical signal sequence and two Ig-likedomains, followed by a probable transmembrane domain and cytosolic tail.The cytosolic tail of the full-length protein contains two tyrosineresidues, of which the first is contained in an ImmunoreceptorTyrosine-based Inhibitory Motif (ITIM; S/I/L/VxYxxL/V). The second is ina motif [NEYSBI] similar, but not identical to the binding site on SLAM(Signaling Lymphocyte Activating Molecule) for SLAM-associated protein(SAP). Nucleotide identity with other human Siglecs (first 750nucleotides) are: Siglec-1, 45.9%; Siglec-2, 48.0%; Siglec-3, 63.6%;Siglec-4, 45.4%; Siglec-5, 64.0%; Siglec-6, 63.0% and many of theresidues typical of the Siglecs are conserved. Siglec-7 forms a closelyrelated cluster with Siglecs 3, 5, and 6.

[1875] Prosaposin & Saposin: Purification Procedure: Purified prosaposinand saposins are prepared from their respective cDNAs (O'Brian et alScience) using standard recombinant techniques.

[1876] Prosaposin is purified from the spent media ofSpodopterafrugiperda (Sf9) cells infected with a baculovirus expressionvector (pAC 610) containing a full-length cDNA for human prosaposin asdescribed by Hiraiwa M et al., FASEB J. 6: 969 (1990). Purificationsteps included column chromatography on Con A-Sepharose andreverse-phase HPLC on a C4 column. The purity of the final purifiedpreparation is assessed by SDS/PAGE, immunoblotting, and N-terminalanalysis. Naturally occurring prosaposin is also isolated by the samemethod from human seminal plasma

[1877] Saposins A, C, and D are all isolated in pure form from thespleen of a patient with Gaucher disease as described by Morimoto S etal., Anal. Biochem 190:154-157 (1990). Saposin B is purified from aheat-treated extract of Gaucher disease spleen by a slightly modifiedprocedure involving reverse-phase HPLC on a C4 column and isoelectricfocusing with the Rotofor cell. Each purified protein gives a singleband on SDS/PAGE, and no cross-contamination of one saposin with anotheris detected after immunoblotting using monospecific antibodies againstsaposins A, B, C, and D, respectively.

[1878] Glycolipid Transfer Protein (GLTP): Purification Procedure:Purified GLTP is isolated and prepared from GLTP cDNA (GTP cDNA) by themethod of Lin et al., J. Biol. Chem. 275:5104-5110 (2000) using standardrecombinant techniques.

[1879] The method of Sasaki T et al., Methods in Enzymology 179: 559-567(1989) is used to purify GLTP to homogeneity with a yield of 19%. Thedata presented derive from the processing of 3,950 g of pig brain (44brains).

[1880] Step 1: Postmitochondrial Supernatant. Fresh pig brains arestored at −20°; thin blocks after removal of pia mater. Frozen brains(4,000 g) are partly thawed by allowing to stand at 20° for 1 hr andthen cut in small pieces, after which all manipulations are performedat, 4°, A 30% homogenate in buffer A is prepared by homogenizing with aWaring blendor for 1.5 mm at high speed. The mixture is centrifuged at8,700 g for 30 mm. The pellet is reextracted by stirring with 10 litersof buffer A. Phenylmethylsulfonyl fluoride (PMSF), 0.2 M in 2-propanol,is added to the postmitochondrial supernatant to a final concentrationof 0.5 mM.

[1881] Step 2: Ammonium Sulfate Precipitation. Ammonium sulfate is addedto the postmitochondrial supernatant to 35% saturation (19.4 g/100 ml).After stirring for 1 hr, the precipitate is removed by centrifugation at8,700 g for 40 mm. The supernatant is adjusted to 70% saturation withammonium sulfate (21.8 g/100 ml) to precipitate GLTP. After allowing tostand overnight, the clear supernatant above the thick precipitate issiphoned off by the use of a tube. The precipitate is collected bycentrifugation (8,700 g for 30 mm), dissolved in 200 ml of buffer C,divided into three dialysis bags (4 cm flat diameter), and dialyzed for2 days against 7 times 4 liters of buffer C. After addition of PMSF to0.5 mM the dialysate is centrifuged at 100,000 g for 2 hr to obtain amembrane-free solution.

[1882] Step 3: First Phosphocellulose Chromatography. The supematant isdiluted to 670 ml with buffer C and applied to a column (7×25 cm) ofphosphocellulose (Whatman P11) in buffer C at a flow rate of 100 ml/hr.Unbound protein is eluted by rinsing the column with 5 liters of bufferC. The transfer activity is eluted with 2 liters of 0.3 M sodiumchloride in buffer C. PMSF is added to the effluent at 0.5 mM.

[1883] Step 4: Chromatography on CM-Cellulose. Protein is precipitatedfrom the effluent with ammonium sulfate added to 90% saturation (60.3g/100 ml), left overnight, and collected by centrifugation at 33,000 gfor 10 mm. The pellets are dissolved in 90 ml of buffer C and dialyzedagainst 4 times 1 liter of buffer C. The dialysate is centrifuged at100,000 g for 30 mm to remove a small amount of insoluble material. Thesupernatant is applied to a column (2.7 )<45 cm) of CM-cellulose(Whatman CM-52 microgranular) equilibrated with buffer C. The column isrinsed with 1.4 liter of buffer C to elute unbound protein. The elutionis continued with a 1.6-liter linear gradient of sodium chloride from 0to 0.2 M in buffer C at a flow rate of 37 ml/hr. Fractions of 17 ml eachare collected, and 50-ul aliquots are assayed. A major peak of transferactivity appears in the effluent at about 55 mM sodium chloride, butsome transfer activity is eluted before the main peak. Fractions of themajor activity peak with an activity of 0.14 nmol of[³H]galactosylceramide transferred/50 ul/30 mm are pooled, and PMSF isadded to 0.5 mM.

[1884] Step 5: Fractionation on Sephadex G75: Protein is precipitatedfrom the pooled fractions with ammonium sulfate (65 g/t00 ml), leftovernight, and collected by centrifugation at 25,000 g for 15 mm. Thepellets are dissolved in 7 ml of buffer B and dialyzed against 5 times300 ml of this buffer. The dialysate is centrifuged at 100,000 g for 90mm, and the supernatant is applied to a column (2×140 cm) of SephadexG-75 (fine, Pharmacia). Protein is eluted with buffer B at a flow rateof 39 ml/hr. Fractions of 4 ml each are collected, and IS-₄ul aliquotsare assayed. Transfer activity appears at VJ V₃=1.84. Fractions with anactivity of 0.17 nmol of [³H]galactosylceramide transferred/15 ul/30 mmare pooled.

[1885] Step 6: Chromatography on Phenyl-Sepharose. Sodium chloride isadded to the pooled fractions to 3 M, which is then applied at a flowrate of 48 ml/hr to a column (2×19 cm) of Phenyl-Sepharose CL-4B(Pharmacia) equilibrated with buffer E containing 3 M sodium chloride.The column is rinsed with 220 ml of buffer E containing 3 M sodiumchloride and 775 ml of buffer E. The transfer activity is eluted with250 ml of 50% (v/v) ethylene glycol in buffer E. Fractions of 4.3 mleach are collected, and 2-ul aliquots are assayed. Fractions with anactivity of 0.03 nmol of [³H]galactosylceramide transferred/2 ul/30 mmare pooled. Extensive washing of the column with buffer E elutes aportion of the transfer activity. Therefore, it is important todetermine the proper time to switch from buffer E to 50% (v/v) ethyleneglycol in buffer E.

[1886] Step 7: Second Phosphocellulose Chromatography The pooledfractions are diluted 2-fold with buffer D and then applied to a column(1.4×15 cm) of phosphocellulose (Whatman P11), which has beenextensively washed and equilibrated with buffer D. The column is rinsedwith 180 ml of buffer D. The elution is continued with a 200-ml lineargradient of sodium chloride from 0 to 0.2 M in buffer D at a flow rateof 20 ml/hr. Fractions of 3.4 ml each are collected, and 2-ul aliquotsare assayed. Most of the transfer activity and protein are eluted in asingle peak at about 93 mM sodium chloride. GLTP in this peak ishomogeneous and has a specific activity of 40.6 nmol/min/mg proteinunder the conditions of [³H]galactosylceramide transfer assay and of 140nmol/min/mg protein under the conditions of PyrGalCer transfer assay. Asmall amount of the transfer activity is eluted as a separate peak afterthe elution of the major peak. The purity of GLTP in the second peakvaries depending on the extent of rinsing with buffer E in Step 6. Nodifference has been found in the properties of GLTP in the first andsecond peaks.

[1887] In vivo Use of Lip-TAA Binding Proteins.: Tumor models given inExamples 18, 20-23 are used. Lip-TAA receptor mimic and Lip-TAA receptorantisense constructs are injected in doses of 0.05 to 50 μg in 0.1-0.5ml of normal saline on days 3, 5, 7, and 9 after the day of subcutaneoustumor implantation or intravenous injection of tumor to establishpulmonary metastases. Control animals are injected with PBS on the sameschedule. The diameters of tumors are measured daily with calipers. Thesizes of the tumor are expressed as the products of the longest diametertimes the shortest diameter (in mm²).On day 21, the mice are killed andeither primary tumors measured or metastatic pulmonary nodules counted.Unless indicated otherwise above, treatments are given one to threetimes per week. The results shown in Example 21 (Table VI) are for eachcomposition and dose tested. The results are statistically significantby the Wilcoxon rank sum test. The other models given in Example 20 arealso suitable for evaluation of the therapeutic effectiveness of theeffector T cells.

[1888] Adoptive Therapy of Tumors in Humans Using Lip-TAA ReceptorDepleted Immunocytes or Antigen Presenting Cells.

[1889] Eligible patients are treated with tumor antigens such asirradiated tumor cells or GM-CSF transduced tumor cells injectedapproximately 10 centimeters from a draining lymph node site. Ten dayspost injection, draining lymph nodes are obtained in a limited surgicalprocedure at the site draining the injection. The lymph nodes areconverted to a single cell suspension of lymphocytes. These lymphocytesare depleted of ganglioside receptors by methods given above. They arethen incubated with various SAg preparations for two days followed byIL-2 for an additional 72 hours. These lymphocytes now called effector Tcells or NKT cell are used for adoptive immunotherapy. Effectorimmunocytes are harvested by centrifugation at 500×g for 15 mm and thecell pellets are pooled. After washing the cells in HBSS, the cell areresuspended in 200 ml of normal saline containing 5% human serum albuminand 450,000 IU of IL-2 for transfer. Each recipient will receives fourescalating doses or 33 million, 100 million, 330 million and 1 billioncells per square meter of body surface area each given one week apart.Cells are infused through a subclavian central venous catheter over a30-minute interval. IL-2 administration iv. is optional and if used isgenerally commenced shortly after completion of cell infusion at a doseand schedule of 180,000 IU/ml every 8 h. for 5 days. All patientsreceive indomethacin (50 mg P.O.) every 8 h, acetaminophen (650 mg P.O.)every 6 h. and ranitidine (150 mg P.O.) every 12 h while receiving IL-2in order to reduce febrile and gastric side effects. As controls, acohort of patients is treated with the in vivo tumor vaccination stepand IL-2 without the tumor effector cells. Patients are followed forclinical response every 4 weeks for 2 months with repeat radiologicalexaminations. Results are given in Example 23 (Table VIII).

EXAMPLE 59

[1890] Preparation and Use of Lip-TAA Receptor Mimics for Inactivationof Lip-TAAs in Tumor Bearing Hosts

[1891] Lip-TAA Isolation: The cellular gangliosides are isolated fromFBL-3 tumor cells which are pelleted lyophilized and extracted twicewith chloroform-methanol (1:1). The total lipid extract was taken todryness. Gangliosides are purified by partitioning the dried totallipids in diisopropylether/1-butanol/0.1% aqueous NaCl. The lyophilizedfinal aqueous phase is dissolved in a small volume of water, after whichSephadex G-50 gel exclusion chromatography is used to remove salt andlow m.w. impurities from the ganglioside fraction. Gangliosides arequantified as nanomols of lipid-bound sialic acid by a modifiedcolorimetric resorcinol assay. Analysis of gangliosides is performedusing 10-×20-cm precoated Silica Gel 60 high performance TLC plates. Theplates are developed in chloroform-methanol. 0.25% aqueous CaCl₂—H,O(60:40:9). Gangliosides are visualized as purple bands withresorcinol-HCl.

[1892] High performance liquid chromatographic purification of Lip-TAA:Lip-TAAs are purified by normal phase HPLC. Briefly, 800 nmollipid-bound sialic acid (LBSA) of FBL-3 cell gangliosides in 100 μl HPLCwater are chromatographed with a Perkin-Elmer (Norwalk. Conn.) HPLCsystem, on a LiChrosorb-NH₂ column (250 mm long, 10 mm inside diameter,Merck, Germany). The separation is conducted at ambient temperature witha solvent gradient program described by Gazzotti et al. (27). Theeluting solvent system is composed of acetonitrile-5 mM Sorensen'sphosphate buffer (83:17), pH 5.6 (solvent A), and acetonitrile-20 mMSorensen's phosphate buffer (1:1), pH 5.6 (solvent B). The gradientelution program of 80 mm, at a flow rate of 6.25 ml/mm, is as follows:100% of solvent A for 7 mm, then a linear gradient from 100% solvent Ato solvent A-solvent B (66:34) over 53 mm, and then a linear gradientfrom solvent A-solvent B (66:34) to solvent A-solvent B (36:64) over 20mm. The elution profile is monitored by flow-through detection of UVabsorbance at 215 nm. The isolated gangliosides are lyophilized,dissolved in a small volume of distilled water, desalted by SephadexG-50 gel exclusion chromatography, and lyophilized.

[1893] Mass spectrometric analysis: The carbohydrate structure of TGGsis characterized by negative-ion fast atom bombardment mass spectrometry(FAB-MS). Ceramide structures are elucidated by negative ion FABcollisionally activated dissociation tandem MS (FAB CAD-MSIMS) withoutprior derivatization using linked scans. Approximately 1 μl ofganglioside-methanol solution is mixed with 2 μl of triethanolamine(matrix) on the FAB probe tip. Ions are formed by bombardment with a6-keV beam of xenon atoms in a JEOL HX-110 double-focusing massspectrometer. For analysis of the ceramide structure by FAB CAD-MS/MSthe [cerO]⁻ fragment ion is selected as the precursor ion. Helium isused as the collision gas, and the helium pressure is adjusted to reducethe abundance of the precursor by 75%. A JEOL DA-500 data systemgenerated the linked scans.

[1894] Lip-TAA Shedding: FBL-3 tumor cells are metabolically labeled byculture for 48 h in RPMI 1640 medium containing 10 MCi each ofD-[1-¹⁴C]glucosamine hydrochloride and D-[1-¹⁴C]galactose, after whichthe cells are washed three times in RPMI medium and resuspended at2.0×10⁶ cells/ml in three 25-cm² tissue culture flasks. The supernatantand the cells from the same flask are harvested 24 h after the cells areresuspended. The gangliosides present in the cells and in the supematantare then purified. The radiolabeled gangliosides are quantified byp-scintillation counting of the ganglioside-associated radioactivity,and equal aliquots of radiolabeled gangliosides are qualitativelyanalyzed by high performance TLC (HPTLC) autoradiography (29) withradiolabeled rat brain gangliosides as standards.

EXAMPLE 60 Construction of Adenovirus Vectors with Insertions forSuperantigens

[1895] Superantigens are inserted into human adenoviruses (Ads) whichare used as live viral vector for expression of superantigens inmammalian cells. Adenoviruses vectors are exemplified here for insertionof the superantigen nucleotide. A mutant adenovirus with selectivity forP53 deficient tumors is preferred such as ONYX-015. An efficient andflexible system for construction of adenoviral vector with insertions ordeletions in early regions 1 and 3 as described by Bett A J et al.,Proc. Natl. Acad. Sci. 91: 8802-8806 (1994) is given below. Similarprocedures insertion of the superantigen gene would be applied to theONYX-014 mutant.

[1896] Principle of Method: Superantigen genes are inserted intoadenoviral vectors using the following principles and methods adaptedfrom Bett, A J et al., Proc. Nat. Acad. Sci. 91: 8802-8806 (1994).Additional methods are given in a book titled Adenovirus Methods andProtocols Wold, WSM ed. Humana Press, Totowa, N.J. (1999) which isincorporated in entirety by reference. These methods involve insertionof the superantigen DNA either by overlap recombination or by ligationinsertion. The method exemplified below for insertion of SAg sequencesuses the Ad5DNA virus but may be adapted to the dl1150 or ONYX-015mutant or any other adenovirus. The Ad5 DNA sequences are cloned intointo bacterial plasmids. Deletions are made in the early region 1and(3180 bp) and early region 3 (2690 or 3132 bp) and are combined in asingle vector that have a capacity for inserts of up to 8.3 kb, enoughto accommodate the majority of cDNAs encding proteins with regulatoryelements. SAg genes are inserted into either early region 1 or 3 or bothand mutations or deletions are readily introduced into the viral genome.

[1897] SAg genes may be inserted into areas of the viral genome thathave been inactivated or deleted and considered to be non-essential tothe lytic activity of the virus or its ability to evade the host immuneresponse. Both Ad and HSV carry genes that are not essential for viralreplication and these may be utilized for SAg insertion.

[1898] The first step is the construction of AdBHG, a virus thatcontains the Ad5 genome with the deletion of E3 sequences from bp 28,133to 30,818 and the insertion of a restriction enzyme site. The next stepis the the generation of a bacterial plasmid containing the entire AdBHGgenome and subsequent identification of infectious clones. Baby ratkidney (BRK) cells are infected with AdBHG under conditions that resultin the generation of circular Ad5 genomes. At 48 h after infection, DNAis extracted from the infected BRk cells and used to transform E. coliHMS174 to ampicillin and tetracycline resistance. Plasmids with thecomplete AdBHG genome are selected. The final step is the generation ofthe pBHG10 by deleting the packaging signals in pBHG9 by partial BamHIdigestion and religation. A Pac I restriction enayme site unique to thisplasmid is present between Ad5 bp 28,133 and bp 30,818 to permit foreigngene insertion. Because the packaging signal is deleted, pBHG10 isnon-infectious but cotransfections with plasmids that contain theleft-end Ad5 sequences including the packaging signal produce infectiousviral vectors with an efficiency comparable to that obtained with pJM17.

[1899] Use of the pBHGE3, pBHG10, or pBHG11 combined with the 3.2-kbdeletion in E1 permits superantigen DNA inserts of ˜5.2,˜7.9, and ˜8.3.respecitvely, into viral vectors. To test the capacity of the BHGsystem, a 7.8 kb consisting of the lacZ gene driven by the HCM promoter(E1-antiparallel orientation) and the SEB gene driven by the beta actinpromoter (E1-parallel orientation) are inserted into the 3.2-kb E1deletion. The 7.8-kb insert is constructed by inserting the 4.1-kb Xba Ifragment from the SEB gene containing the SEB gene driven by the betaactin promoter into the Xba I site in pHCMVsp1LacZ generating pHlacSEB.The isolate pHlacSEB expressed both lacZ and SEB at levels comparable tothose obtained with vectors containing single inserts.

[1900] The Method: The first step involves the construction of AdBHG, avirus that contains the Ad5 genome with the deletion of E3 sequencesfrom bp 28,133 to 30,818 and the insertion of modified pBR322 at bp1339. AdBHG is made by cotransfection of 293 cells with purified viralDNA from Ad5PacI, digested with Cia I and Xba I, and pWH3.

[1901] The next step involves the generation of a bacterial plasmidcontaining the entire AdBHG genome and subsequent identification ofinfectious clones. Baby rat kidney (BRK) cells are infected with AdBHGunder conditions that result in the generation of circular Ad5 genomes.At 48 h after infection DNA is extracted from the infected BRK cells andused to transform E. coli HMS 174 to ampicillin and tetracyclineresistance (Apr and Tetr, respectively). From two experiments, plasmidDNA from a total of 104 colonies is screened by HindIII and BamHI/Sma Idigestion and gel electrophoresis. Plasmids that appear to posses acomplete AdBHG genome are selected and all four are found to beinfectious when transfected into 293 cells.

[1902] The final step involves generation of pBHG10 by deleting thepackaging signals in pBHG9 by partial BamHI digestion and religation.The left and right termini of the Ad5 genomes are covalently joined anda segment of plasmid pBR322 is present between AdS bp 188 and 1339 toallow propagation of pBHG10 in E. coli. A Pac I restriction enzyme site,unique in this plasmid, is present between AdS bp 28,133 and bp 30,818to permit insertion of the superantigen genes. Because the packagingsignal is deleted, pBHG10 is noninfectious but cotransfections withplasmids that contain the left-end AdS sequences including the packagingsignal produce infectious viral vectors with an efficiency comparable tothat obtained with pJM17.

[1903] To generate two useful variants, pBHGE3 and pBHG11 areconstructed from the original plasmid pBHG10. pBHGE3 permitsconstruction of vectors with wt E3 sequences and pBHG11 increase thecloning capacity of resulting viral vectors. The 2.69-kb E3 deletion inpBHG10 removes the major portions of all E3 mRNAs, the first E3 3′splice acceptor site, and the L4 polyadenylylation site but leaves theE3 promoter, the 5′ initiation site, the first E3 5′ splice donor site,and the E3b polyadenylylation site intact. Viruses with the 2.69-kb E3deletion have the same growth kinetics and progeny virus yields as wtvirus. The 3.1-kb E3 deletion in pBHG11 removes two additional elementsnot removed by the 2.69-kb E3 deletion: the first E3 5′ splice donorsite and the E3b polyadenylylation site. This deletion does notinterfere with the open reading frame for pVIII or any of the L5 familyof mRNAs. Viruses containing the 3.1-kb deletion give wt progeny yieldsin infected 293 cells.

[1904] To maximize the capacity of the BHG system and to facilitate theintroduction of inserts such as the SEB gene into the E1 region,plasmids containing a 3.2-kb deletion of E1 sequences and multiplerestriction sites for the insertion of foreign genes have beenconstructed. This deletion leaves intact the left ITR and packagingsignals and extends just past the Spi binding site of the protein IXpromoter. The promoter for transcription of the protein IX gene isrelatively simple, consisting of this Spi binding site and a TATA box.The Spi binding site is essential for expression of protein IX and it istherefore, reintroduced at a position 1 bp closer to the TATA box thanin the wt promoter. However, neither the original 3.2-kb E1 deletion northe deletion mutants containing the synthetic Spi site are significantlyaltered in protein IX expression, heat stability or final progeny yieldsof viruses with this deletion.

[1905] General Treatment Plan for Patients with the SAg-dl1150Construct: SAg-dl1150 is administered intratumorally to patients withrecurrent and refractory cancers. The efficacy of SAg-dl1150 treatmentis based on the injected tumor(s) response. The clinical benefit ofSAg-dl1150 is evaluated through quality-of-life assessment (EORTCinstrument), Karnofsky performance score, and pain assessment. Survivaland progression-free survival intervals are recorded. Results are givenin Example 23 (Table VIII).

[1906] SAg-dl1150 Dosages and Dosing Rationale: Patients are treatedwith SAg-dl1150 administered daily for 5 d at a dose of 10¹⁰ pfu perday. This is the highest dose administered daily for 5 d in the phase Istudy and was shown to be safe (i.e., no dose-limiting toxicities).

[1907] Treatment with SAg-dl1150:

[1908] a. Dosing Regimen: For administration of each dose of, patientsare treated and observed in a properly equipped outpatient clinic. Thetarget tumor is injected with 10¹⁰ PFU of SAg-dl1150 daily over 5 d(i.e., a total dose 5×10¹⁰ PFU) (with day 1 being the first day ofSAg-dl1150 injection. Nontarget tumor(s) (where applicable) are injectedwith either diluent or SAg-dl1150 on the same days in identical fashionto the target tumor following the guidelines detailed in steps below.

[1909] b. Target Tumor Masses: The dominant, symptom-causing tumor (ifsymptoms are present) is identified as the target tumor and is the onlytumor injected with SAg-dl1150 during the first two treatment cycles.The identification of the most symptomatic, problematic lesion is basedon the judgement of the Principal Investigator. Multinodular, butcontiguous tumors are treated and evaluated as a single lesion.

[1910] c. Secondary, Nontarget Tumor Masses: If additional, smaller,accessible lesions are present, these lesions are injected with diluentfor the first two treatment cycles as described in step 3 below.Thereafter, treatments are divided between up to three separate lesions(i.e., the initial two cycles are concentrated within the dominantlesion; thereafter, 6 wk after treatment initiation, two additionalsecondary lesions are injected). However, the total dose to the patientremains the same (i.e., the same total dose will be divided up betweenthe tumors to be treated); the total volume in which the SAg-dl1150 issuspended will be increased based on the total tumor volume of thetumors to be treated. If a CR occurs in a treated lesion, injections canbe continued as outlined above with newly defined dominant and secondarylesions.

[1911] d. Immediate Posttreatment Monitoring of Patients: The patient'svital signs are taken ≦15 mm before each SAg-dl1150 injection. Aftereach injection is completed, the patient will be observed in the clinicfor a minimum of 30 mm. Vital signs are taken after 30 min ±5 mm. Ifvital sign(s) have changed by >15%, vital signs will be repeated every30 mm until returning to within baseline 15% of baseline values.Following the observation period, the patient is sent home orhospitalized overnight at the discretion of the investigator. Resultsare given in Example 23 (Table VIII).

[1912] Plasmid Superantigen-HPV E7 Constructs.: The method of Chen CIIet al., Cancer Res. 60 1035-1042 (2000) is used. A DNA fragment encodingSEB is prepared as described in Example 1 and Dow S W et al., J. Clin.Invest. 101: 2406-1414 (1998). Briefly, the genes for SEA and SEB andthe pHBNeo beta actin promoter which are to be inserted into the BHGsystem at multiple restriction sites are described in Example 1Additionally the SEB and SEA genes are obtained from Dr. John Kappler(National Jewish Hospital), and the gene for TSST-1 are obtained fromDr. Brian Kotzin (National Jewish Hospital). After PCR amplification,all three genes are subcloned into a eukaryotic expression vector (PCR3;Invitrogen Corp., San Diego, Calif.). The primers used for cloning SEAwere (forward): GGGAATTCCATGGAGAGTCAACCAG; (backward):GCAAGCTTAACTTGTTAATAG; for SEB (forward): GGGAATTCCATGGAGAAAAGCG;(backward): GCGGATCCTCACTTTTTCTTTG; for TSST-1 (forward):CGGGGTACCCCGAAGGAGGAAAAAAAAATGTCTACAAACGAT AAT ATAAAG; (backward):TGCTCTAGAGCATTAATTAATTTCTGCTTCTATAGTTTTTAT.

[1913] The full-length TSST-1 gene is cloned into PCR3, whereas only themature SEB and SEA genes (minus the putative bacterial signal sequences)are cloned into PCR3. Removal of the SEB and SEA signal sequencesincreased the level of gene expression in transfected cells (data notshown). The plasmids are grown in E. coli and plasmid DNA used forrecombination in the adenoviral constructs are extracted by modifiedalkaline lysis-method and purified on a CsCl gradient. For thegeneration of SEB-expressing plasmid, (pcDNA-SEB) the SEB was subclonedinto the unique BamHI and HinIII cloning sites of the pcDNA3.1(−)expression vector (Invitogen, Carlsbad, Calif.) downstream of thecytomegalovirus promoter. For the generation of HPV-16 E7-expressingplasmid (pcDNA3-E7), E7 DNA is amplified by PCR using primers designedto generate BamHI and HindIII restriction sites as the 5′ and 3′ ends ofthe amplified fragments, respectively. The amplified E7 DNA is thencloned into the unique BamHI and HindIII cloning sites of the pcDNA3.1.For the generation of E7-SEB chimera (pcDNA-E7-SEB), E7 DNA is amplifiedby PCR using primers designed to generate BamHI restriction sites atboth 5′ and 3′ ends of the amplified fragments. The E7 DNA is thensubcloned to the 5′ end of pcDNA3-SEB. The accuracy of these constructsis confinned by DNA sequencing. Plasmid DNA with SEB, E7, or E7-SEB geneinsert and the “empty” plasmid vector are transfected into subcloningefficient DH5 (TM cells; Life Technologies). The DNA is then amplifiedand purified using double cesium chloride purification (BioServeBiotechnologies. Laurel, Md.). The integrity of plasmid DNA and theabsence of Escherichia coli DNA or RNA are checked in each preparationusing 1% agarose gel electrophoresis. DNA concentration os determined bythe absorbance measured at 260 run. The presence of the inserted E7fragment is confirmed by restriction enzyme digestion and gelelectrophoresis.

[1914] DNA Vaccination.: Gene gun particle-mediated DNA vaccination isperformed using a helium-driven gene gun (Bio-Rad. Hercules, Calif.)according to the protocol provided by the manufacturer. Briefly,DNA-coated gold particles were prepared by combining 25 mg of 1.6˜m ofgold microcartiers (Bio-Rad, Hercules, Calif.) and 100 M¹ of 0.05 Nispermidine (Sigma, St, Louis, Mo.). Need to house this chimeric plasmidconstruct in a viral vector which can transport the chimeric DNA intotumor cells for transport selectively to tumor cells.

[1915] Generation of Dendritic Cells Exposed to Necrotic Tumor CellsTransfected with: Ag Genes: DCs are generated from blood monocyteprecursors as follows. Briefly, mononuclear cells are plated in 6-wellculture plates at 8×10⁶ cells per well and allowed to adhere to plasticfor 2 hours. Plastic adherent cells are cultured in RPMI-1640(BioWhittaker, Walkersville, Md., USA) supplemented with 1% autologousplasma, 20 μg/ml gentamicin (clinical grade; Fujisawa USA, Deerfield,Ill., USA), 800 U/mL GMP grade IL-4 (Cell Genix, Freiburg, Germany), and100 IU/mL clinical grade GM-CSF (Immunex, Seatde, Wash., USA) for 7days. Cultures are supplemented with cytokines on days 2,4, and 6. Onday 7, cells are transferred to new plates and cultured in the presenceof 50% (vol/vol) monocyte-conditioned medium (MCM) for 2 additionaldays. Using this methodology, 1.5×10⁶ to 4×10⁶ mature DCs expressinghigh levels of CD86, CD83, and HLA-DR, and lacking other lineage markers(e.g., CD 14), are reproducibly generated from 80 ml of peripheralblood. MCM is prepared by adhering 50×10⁶ PBMCs in 7 ml of 1% autologousplasma to plates precoated with 100 μg/mL human IgG (Bayer Corp.,Elkhart, Ind., USA) for 90 minutes. The PBMCs are harvested afterovernight culture of adherent cells and are filtered and frozen at −20°C. before use. Mature DCs generated using this methodology are potentstimulators in mixed lymphocyte reaction at T/DC ratios of 900:1.

[1916] Injection of SAg-Tumor Antigen Pulsed Dendritic Cells: Antigensare added to DCs overnight 1 day before injection. On the day ofinjection (i.e., day 9 or 10 of culture), the DCs are washed free ofantigen and resuspended in normal saline containing 0.5% autologousplasma in two 0.2 ml aliquots. Phenotype and purity of injected DCs aremonitored by the expression of the DC maturation-associated marker CD83by flow cytometty. DCs are injected as a superficial subcutaneousinjection in 2 adjacent sites on the upper inner arm, ˜4 inches from theaxilla. All injected DC preparations test negative for bacterial andfungal contamination. All subjects are observed overnight. Results aregiven in Example 23 (Table VIII).

[1917] Viral Vector Containing Superantigen and IκBα Nucleic Acids.: Thesuper-repressor IκBα and superantigen nucleotides are cloned into thereplication-deficient Ad5 vector with transgene expression driven by theCMV pormoter. The replication deficient Ad.CMV3 containing thesuperantigen and super-repressor IκBα is used ot produce stablytransfected HT10801 cells. The cells are pre-infected with Ad.CMV IκBα;or Ad.CMV3 empty control vector for 1 h.

[1918] In vivo tumor model with Ad.CMV IκBα and TNF treatment.: HT1 0801cells (10⁶ cells/0.1 ml) or control HT1080V cell suspensions areinjected subcutaneously into each flank of female, athymic, nu/nu mice5-6 weeks old (Taconic, Germantown, N.Y.). Tumors are allowed to developfor 21 d and then were injected with TNFα; (10 μg/100 μl in PBS once aday for 3 d). Both maximum and minimum diameters of resulting tumor aremeasured over the skin every 2 days using a slide caliper. Tumor cells(10⁶) were injected subcutaneously in the flanks of nu/nu, female,athymic nude mice 5-6 weeks old (body weight, 18-20 g). Tumors aretreated 2-3 weeks after cancer cells are injected when tumor dimensionswere approximately 1 cm×1 cm. Tumor treatment involved the intratumoralinjection of recombinant adenovirus Ad.CMV Iκ Bα. Virus (10¹⁰ PFU,diluted in a total of 200 ul of PBS) is administered in a single pass ofa 27-gauge hypodermic needle. A second virus injection was given 7 dafter the first. The recombinant adenovirus Ad.CMV3 and vehicle aloneare used as controls. CPT-11 (Irinotecan; Pharmacia & Upjohn Company,Kalamazoo, Mich.) was administered by tail vein injection at a dose of33 mg/kg in 100 ul every 4 d beginning 24 h after the first viralinjection. A total of five doses of CPT-11 are given. PBS (100 ul)administered by tail vein injection was used as a control for drugtreatment. Tumor diameter is measured along the longest orthogonal axesevery other day after treatment was initiated, and recorded as tumorvolume. Tumor volume is calculated by assuming a spherical shape, usingthe formula (volume=4/3 πr²), where r=1/2 (mean tumor diameter measuredin two dimensions). Tumors are fixed in 10% formalin and embedded inparaffin blocks. Paraffin-embedded sections are pre-treated with dewaxand rehydrated (55° C. for 15 mm), then washed in xylene and rehydratedthrough a graded series of ethanol and redistilled water. Tissuesections were then incubated with proteinase K (20 μg/μl in 10 mMTris/HCl, pH 7.4-8.0, for 15 mm at 21-37° C.), permeabilized in 0.1%Triton-X100 in 0.1% sodium citrate, and then labeled with the TUNELreaction mixture (Boehringer). Results are given in Example 21 (TableVI).

[1919] Therapeutic Model: The therapeutic efficacy of SAg-dl1520construct is determined in vivo by growing p53-C33A cells and p53+U87human glioblastoma multiforme cells as tumor xenografts in athymic mice.The tumor cells are injected subcutaneously into each flank of eachmouse, and after establishment of palpable tumors (mean tumor volume 150μl), the tumors are directly injected with CsCl-purified wild-typeadenovirus, with SAg-dl1520. dl1520 or ultraviolet (UV)-inactivatedwild-type virus as a negative control every other day for three totaldoses. Tumor growth is followed for 6 weeks, at which time the meantumor volume in each group are determined. Treatment of C33A tumors withsuperantigen-dl1520 results in a 100% reduction in mean tumor volume ascompared with dl1520 cells or wild type UV inactivated adenovirus(unpaired two tailed test: P=0.02). In contrast, dl1520-injected p53+U87 tumors are comparable in size to control-injected tumors after 6weeks, although shrinkage is seen in some cases.

[1920] Construction of plasmids encoding SAg self replicating RNA(pRep-SA): pRep-SAg is constructed by replacing the LacZ fragment inpRep-LacZ with SAg fragment from the plasmid PHBNeo-SEB or by using theplasmids developed by Dow as in the above section on viral DNA.pRep^(del)-LacZ is created by cleavage of the Rep-LacZ plasmid with XhoIand BgIII (creating a 1407-bp deletion) which was then end-filled andself-ligated.

[1921] The plasmid SFV3-LacZ contains a SP6 promoter, a 7-kb fragmentencoding the SFV RNA replicase, and a subgenomic promoter that is boundby the RNA replicase to synthesize large quantity of subgenomic RNA. TheLacZ gene is located in the 5′ region of the subgenomic promoter. Theplasmid pRep-LacZ is constructed by inserting an SphI fragmentcontaining the CMV immediate early promoter/enhancer into the SphI siteof SFV3-LacZ (Life Technologies). The CMV promoter/enhancer fragment isinserted so that the resulting plasmid could potentially be used for DNAimmunization studies similar to those reported. An additional SpeI siteis inserted 5′ of the 5P6 promoter together with the CMVpromoter/enhancer fragment. Digestion of the plasmid with SpeI generatesa DNA fragment that contains only the SFV RNA replicase and β-gal codingsequences without sequences encoding ampicillin-resistant p-lactamase,which is used as the template for in vitro transcription of Rep-LacZRNA. The primers used for generating a PCR fragment containing the CMVpromoter/enhancer from pCR3.1 plasmid (Invitrogen, Carlsbad, Calif.)are: 5′ primer, 5′-ACATGCATGCACTAGTGCGCGCGTTGACATTGATTA-3′ (SpeI site(underlined) added for subsequent linearization) and 3′ primer,5′-ACATG-CATCCATGTGAGAGCTCTGCTTATATAGACC-3′.

[1922] Intravenous tumor model: BALB/c mice are immunizedintramuscularly with RNA vaccines, then 21 d after immunization, micewere injected intravenously with 5×10⁵ CT26.CL25 cells. Then 12 d later,pulmonary metastases are counted by experimenters ‘blinded’ to sampleidentity. For experiments with established CT26.CL25 tumor, BALB/c miceare injected intravenously with 10⁵ CT26.CL25 cells, and tumor isallowed to establish itself for 2 d. Mice are then therapeuticallyimmunized with 100 μg RNA, then assessed for survival. Alternatively,0.1 μg IL-12 (Genetics Institute, Cambridge, Mass.) is administeredintravenously once daily for 3 d starting 12 h after the therapeuticimmunization and survival is assessed. Results are given in Example 21(Table VI).

EXAMPLE 61 Isolation of Lipid Rafts

[1923] Lipid rafts were isolated using modified lysis conditions andflotation on discontinuous sucrose gradients (20, 36, 37). In brief,cells (10⁸) are washed with ice-cold PBS and lysed for 30 mm on ice in1% Triton X-100 in TNEV containing protease and phosphatase inhibitors(TNEV: 10 mM Tris/HCl, pH 7.5, 150 mM NaCl, and 5 mM EDTA; CLAP [2.5mg/ml each of chymostatin, leupeptin, antipain, and pepstatin Λ in DMSO]and 1 mM sodium orthovanadate). The lysis solution is furtherhomogenized with ten strokes in a Wheaton loose-fitting douncehomogenizer. Nuclei and cellular debris are pelleted by centrifugationat 900 g for 10 min. For the discontinuous sucrose gradient, 1 ml ofcleared supernatant is mixed with 1 ml of 85% sucrose in TNEV andtransferred to the bottom of a Beckman 14×89 mm centrifuge tube. Thediluted lysate is overlaid with 6 ml 35% sucrose in TNEV and finally 3.5ml 5% sucrose in TNEV. The samples are centrifuged in an SW41 rotor at200,000 g for 16-20 h at 4° C. 1-ml fractions are collected from the topof the gradient.

EXAMPLE 62 Identification and Characterization of StreptococcalPyrogenic Exotoxins, Staphylococcal Enterotoxins and SETs

[1924] SPEA Allelic Forms and Mutants. The method of preparation of SPEAallelic forms and mutuants is carried out by the method of Kline J B etal., Infect. Immun. 64: 861-869 (1996).

[1925] Purification of SPEA from S. pyogenes. One-liter cultures of 5.pyogenes Ros (generous gift of D. L. Stevens, Idaho VA Medical Center)are grown in NCTC-135 medium (Gibco/BRL, Grand Island, N.Y.)supplemented with glucose (21). Toxin was partially purified fromcell-free culture filtrates by differential solubility in ethanol andacetate-buffered saline. Toxin which were precipitated four times wereredissolved in 0.1 M imidazole-acetic acid (pH 5.0) and applied to aQAE-Sephadex A-50 (Pharmacia Fine Chemicals, Uppsala, Sweden) jacketedcolumn. The toxin was eluted as a single peak with a NaCl gradient asdescribed previously. Sodium dodecyl sulfate (SDS)-polyacryl-amide gelelectrophoresis (PAGE) analysis of purified SPEA reveals a single bandwith the expected molecular mass of SPEA (25.8 kDa). The toxin isdiaryzed against phosphate-buffered saline (PBS) and stored at −20° C.

[1926] Construction of pET15b-spea1. 150 ng of plasmid pA2 containingthe SPEA gene (kindly provided by J. J. Ferretti, Oklahoma City, Okla.)is used as a template to amplify a 663-bp DNA fragment by PCR usingprimers 19b-A1 (5′-CCCCATATGCAACAAGACCCCGAT-3′) and 19b-A2(5′-GGGGGATCCTTACTTGGTTGTTAG-3′).

[1927] These primers encode terminal BamIII and NdeI restriction sites,respectively. After digestion with BamHI and NdeI (Gibco/BRL), the DNAfragment, which encodes the mature protein without the leader peptide,is cloned into BamHI- and AMd-digested pET15b (Novagen, Madison, Wis.),producing the construct pET15t<<pe/i7. The complete nucleotide sequenceof the inserted fragment is confirmed by the dideoxy-chain terminationmethod. In .E. coli BL21(DE3) (Novagen), this construct expresses afusion protein consisting of an N-terminal six-histidine-residue tag andSPEA1.

[1928] Generation of point mutations in SPEA. Site-directed mutagenesisof SPEA1 is performed by using PCR with oligonudeotides containing thedesired nucleotide substitution. Briefly, 150 ng of pET15b-speA1, themutant ohgonucleotide, and either primer 19b-A1 or primer 19b-A2 wereused to generate two SPEA fragments with complementary ends. A secondPCR is performed with the two overlapping SPEA fragments and flankingprimers 19b-A1 and 19b-A2 to generate the full-length mutated SPEA gene.This PCR product is then digested with BamHI and NdeI and inserted intopET15b as described above. The complete nucleotide sequences of bothstrands of each mutated SPEA are determined by the dideoxy-chaintermination method to ensure that only the single desired mutation waspresent.

[1929] Recombinant toxin nomenclature. Recombinant SPEA1 (rSPEA1) aminoacid substitution mutants are named according to the original aminoacid, its position in the mature toxin, and the resulting amino acid.For example, for rSPEA1-N₂0A, amino acid residue 20 was changed fromasparagine to alanine. All mutant recombinant proteins generated containsingle amino acid substitutions except for rSPEA1-S51L,N55A andrSPEA1-C87S,C90S, which have two substitutions. rSPEA1 is the toxinencoded by SPEA1 (40). rSPEA2 (also referred to as rSPEA1-G80S) is thetoxin encoded

[1930] Expression and purification of rSPEA. Expression and purificationof the recombinant toxins by using the pET expression vector is asdescribed by manufacturer (Novagen). In brief, E. coli BL21 (DE3) wastransformed with pET15-SPEA constructs for production of recombinanttoxins. In this background, SPEA is under the control of a T7 promoter,and the T7 polymerase gene is on the E. coli chromosome under thecontrol of an isopropylthio-D-galactopyranoside (IPTG)-inducible lacpromoter. Cultures are grown to mid-exponential phase and induced toexpress toxin by the addition of 0.4 mM IPTG (Sigma Chemical Co., StLouis, Mo.). Cultures are grown for an additional 3 h after induction,harvested by centrifugation, and disrupted by sonication. rSPEApreparations are purified by metal chelation chromatography usingHis-Bind resin (Novagen). One hundred to 500 μg of toxin is digestedwith 1 μg of thrombin (Novagen) for 16 h at room temperature. The toxinis then purified from the His-tag leader sequence by ultrafiltrationwith 10,000-moleculat-weight cutoff filters (MSI, Westboro, Mass.). InE. coli BL21(DE3) (Novagen), this construct expresses a fusion proteinconsisting of an N-terminal six-histidine-residue tag and SPEA.

[1931] Generation of polydonal antisera reeognizing SPEA. Female NewZealand White rabbits are by SPEA2. The toxin encoded by SPEA1, SPEA3,is also termed rSPEA1-V761.immunized subcutaneously with 50 μβ ofcommercially available SPEA1 (Toxin Technologies, Sarasota, Fla.) incomplete Freund's adjuvant (Gibco/BRL). Subsequent immunizations of 25mg of toxin are administered at week 3 and then every 2 weeks inincomplete Freund's adjuvant (Gibco/BRL). Sera were first collected atweek 6.

[1932] Western blot (immunoblot) analysis of rSPEA. Each of the mutanttoxins and allelic forms is screened for instability by Westernanalysis. Toxins are analyzed by SDS-PAGE (12% acrylamide) andelectroblotted to nitrocellulose. The nitrocellulose filters areincubated overnight in PBS supplemented with 5% low-fat dry milk andthen stained with polyclonal rabbit antiserum against SPEA1. Anti-SPEAantibody binding is detected with horseradish peroxidase-labeled goatanti-rabbit antibody. Bands were visualized with 4-chloro-1-naph-thol(Sigma).

[1933] SDS-PAGE analysis. To look for the presence of disulfide bondformation between cysteine residues of rSPEA1, 2-p.g aliquots ofpurified toxins are mixed with gel running buffer (50 mM Tris-HCl [pH6.8], 2% SDS, 0.1% bromophenol blue, 10% glycerol) with or without2-mercaptoethanol (final concentration, 1%). The samples are then boiledfor 5 min and electrophoresed for 5 h at 40 mA on an SDS-12%polyacrylamide gel Protein bands were visualized by staining withCoomassie brilliant blue R250 (Bio-Rad, Melville, N.Y.).

[1934] Mitogenicity assays. Heparinized whole blood is obtained fromhealthy donors. Samples were fractionated on Ficoll-Paque (PharmaciaBiotech, Pisca-taway, N.J.), and the peripheral blood mononuclear cells(PBMCs) are harvested and washed three times in PBS. Then cells (10⁵)were added to 96-well U-bottom plates in 200 μl of complete RPMI1640supplemented with 10% fetal calf serum (PCS). PBMCs are incubated for 72h at 37° C. with various concentrations of rSPEA toxins underatmospheric conditions of 5% CO₂; 1 uCi of [3H]thymidine (ICNBiochemicals, Costa Mesa, Calif.) is added to each well, and the cellsare incubated for an additional 24 h. Cells were harvested onto glassfiber filters, and [³H]thymidine uptake is quantitated by liquidscintillation counting. For each mutant toxin, PBMCs from at least threedistinct donors are used.

[1935] Flow cytometry of PBMCs. PBMCs (10⁶) from healthy donors areincubated with toxins at a concentration of 1 μg/ml for 4 days. Cellswere harvested, washed three times with PBS, and applied to a FACScanflow cytometer (Becton Dick-inson).

[1936] Cell lines. L-cell transfectants L66 (vector only) and L54.1(DQβ3/DQα2) are the generous gift of Robert Karr, Monsanto Company.Transfectants were maintained in suspension in petri dishes in Oulbeccomodified Eagle medium (DMEM) with 10% PCS, 2 mM L-glutamine, 100 U ofpenicillin per ml, 100 μg of streptomycin per ml, and 250 μg of theneomycin analog G418 per ml for selection. Before use, transfectants areexamined by fluorescence-activated cell sorting analysis withfluorescein isothiocyanate-labeled anti-HLA-DQ3 (KS13) to confirm theexpression and surface localization of the DQ molecule. Antibody KS13 isthe generous gift of Soldano Ferrone, New York Medical College,Valhalla, N.Y.

[1937] Radiolabeled rSPEA binding assays. rSPEA1 is iodinated by usingchloramine-T (Sigma). One hundred μg of toxin was incubated with 0.5 mCiof Na¹²⁵I and 5 μl(5 mg/ml) of chloramine-T in 100 μl of 100 mM Tris-150mM NaCl (pH 7.4) for 10 min. The reaction is terminated by the additionof 20 μl (5 mg/ml) of sodium metabisulfate (Sigma). Labeled toxin wasseparated from unincorporated radioactivity on a 1-ml Sephadex G-25column, which had been preequilibrated with PBS. The Kd of rSPEA-DQinteraction is determined by incubating 10⁶ L54.1 cells (expressingclass II MHC) with various concentrations of mI-rSPEA in a total volumeof 100 μl of DMEM-10% FCS-0.1% sodium azide. Nonspecific binding isestimated by incubating separate tubes with unlabeled competitor toxinat a concentration 100 times greater than that of labeled toxin. Cellsare incubated at 37° C. for 4 h with agitation every 20 min and thenpelleted through an oil gradient (80% dibutyl phthalate, 20% olive oil).Pellets are cut from the tubes, and cell-associated ¹²⁵I was measured ona gamma counter.

[1938] K, determinations are evaluated in a similar fashion except thatadditional tubes containing various concentrations of ¹²⁵I-rSPEA phisunlabeled mutant competitor are analyzed. Lineweaver-Burk plots of thereciprocal of toxin bound versus toxin free are used to determineinhibition constants.

[1939] Structure of SPEA. Predicted ribbon structure of SPEA wasgenerated by the Swiss Model Automated Protein Modelling Server, GlaxoInstitute for Molecular Biology, Geneva, Switzerland. Primary amino acidsequence of SPEA is modeled on the crystal structures of staphylococcalenterotoxin A (SEA) and staphylococcal enterotoxin E (SEE). Crystalcoordinates for SEA and SEE are from the Brookhaven Database CrystalCoordinates and are deposited by Swaminathan and Sax. Structure isviewed by using the Raswin Molecular Graphics Viewer software, version2.4, 1994 (R. Sayte, Department of Computer Science, University ofEdinburgh, Edinburgh, United Kingdom).

[1940] Isolation and Purification of SSA is by the method of Mollick Jet al., J. Clin. Invest. 92: 710-719 (1993) and Reda K et al., Infect.Immun. 62: 1867-1874 (1994)

[1941] Purification of SSA from S. pyogenes strain Weller. Because RDAhas been used to purify several staphylococcal enterotoxins, thismaterial is useful in identifying novel S. pyogenes superantigens.Concentrated culture supernatants from strain Weller are chromatographedon a RDA column, the column was eluted with a phosphate step gradient,and fractions are tested for the presence of a class II-dependent T cellmitogen. We identify such an activity eluting between 60 and 150 mM PO4,corresponding approximately to fraction numbers 8-55. This activityelutes in a broad peak and does not correspond to a detectable proteinpeak. Examination of an aliquot of the pooled activity by SDS-PAGE gelreveals many proteins, some in the 30-kD range. The pooled activefractions are fractionated from the RDA column by gel filtration (G-75)and anion exchange chromatography and active fractions from each columnare selected. The product from the final chromatography stepconsistspredominantly of three proteins. The proteins are blotted to asolid support and analyzed by NH2-terminal sequencing. The higher M_(r)protein is identified as SP and/or SPE-B. These two proteins are closelysimilar and are not distinguished based on the 29 amino acids sequenced.The lower M_(r) protein, ˜27 kD in size, yields a 59-amino acid NH2terminus that is not notably homologous to any previously characterizedprotein. The middle band (28 kD) displays an NH2 terminus strikinglysimilar to the NH2 termini of SEB, SEC, and SEC3 and dissimilar to Mprotein. The 28-kD molecule with the SEB-like NH2 terminus is designatedSSA.

[1942] Purification of SSA by Ab affinity chromatography. To determinewhether or not SSA isresponsible for the superantigen activity, ourefforts are directed to its purification. An anti-peptide antiserum israised against the first 19 amino acids of SSA. To determine the abilityof the anti-SSA Abs to bind native SSA, a concentrated streptococcalsupernatant from 16 liters is chromatographed on RDA in an effort toenrich for SSA. The RDA eluate is passed over the anti-SSA Ab column andthe column eluted. Examination of the eluate by SDS-PAGE gel, and silverstain shows one prominent band at 28 kD, and two minor bands, one at ˜25kD and one at ˜12 kD. NH2-terminal sequencing of the 28-kD product showsthe SSA NH2 amino terminus. To determine whether the lower M_(r) specieswere contaminants or SSA degradation products, an identical sample issubjected to immunoblot analysis. Anti-SSA antibodies detect all threespecies shown in the silver stain gel, indicating that these lowermolecular weight bands are breakdown products of the 28-kD protein.Because the antibodies are directed against the NH2 terminus, theseproducts likely represent SSA molecules missing COOH terminal sections.

[1943] PCR amplification and cloning of the 5′ half of ssa from Wellergenomic DNA. Nondegenerate, nonoverlapping oligonucleotides (SSA1,5′-AGTCAACCAGATCCTACGCCAG AACAATTGAA-3′; SSA2, 5′-AAATCGAGTCAATTTACGGAGTTATGGCC-3′) are designed on the basis of the SSA N-terminal proteinsequence with a bias toward SEB codon usage. We hypothesized that SSAmay retain homology to SEB in regions further downstream from the 24N-terminal residues, especially in regions relatively conserved amongall known staphylococcal and streptococcal superantigens. In order toamplify ssa from Weller genomic DNA with PCR, we pair each SSAoligonucleotide with an oligonucleotide (SEB7, residues 658 to 675)specific for a region in SEB immediately downstream of the disulfideloop. Weller genomic DNA (200 ng) is combined with 50 pmol each of sense(SSA1 or SSA2) and antisense (SEB7) primers, a 200 μM concentration ofeach deoxynucleoside triphosphate, and 10 μl of 10×Pfu polymerase buffer1 (Stratagene) in a total volume of 100 μl. Reaction mixtures areoverlaid with 100 μl of mineral oil and denatured at 95° C. for 7 minbefore Pfu polymerase (2.5 units) (Stratagene) is added. PCR conditionswere as follows: 1 min at 95° C., 2 min at 37° C., and 3 min at 72° C.for 25 cycles in a thermocycler (Perkin-Elmer Corp., Norwalk, Conn.).Combinations of SSA1 or SSA2 with SEB7 specifically amplified productsof approximately 340 or 310 bp, respectively, from strain Weller genomicDNA, but not from strain Gall DNA, which does not produce SSA, PCRproducts were ligated to the pBluescript SK˜ vector to make pKR1 andpKR2, which are used to transform XL-1 Blue E. coli. Nucleotide sequenceanalysis of pKR1 and pKR2 inserts predicted amino acid sequencesidentical to that determined by N-terminal protein sequencing of nativeSSA from strain Weller S. pyogenes (39).

[1944] Subcloning and expression of recombinant SSA. The nucleotidesequence encoding the mature form of SSA is PCR amplified from Wellergenomic DNA with flanking primers and digested with HincII and SpeI,which cut 46 bp upstream and 33 bp downstream, respectively, of the ssaopen reading frame. This fragment was ligated to the pBluescript II KS˜expression vector (Stratagene) to make pKR4. An XL-1 Blue E. coli straincarrying pKR4 was grown to an optical density at,600 nm of 1.0 andinduced to express SSA by the addition of IPTG(isopropyl-β-D-thiogalactopyranoside) to a final concentration of 0.1mM. After further incubation at 37° C. with shaking for 3 h, bacteriawere harvested by centrifugation and resuspended in TE, pH 8.0. Analiquot of cells was then mixed with an equal volume of SDS samplebuffer, and the whole cell lysate was analyzed by SDS-polyacrylamide gelelectrophoresis (SDS-PAGE) and SSA immunoblot analysis. Lysates of E.coli strains carrying pMV7 or pBluescript, containing seb or no insertin the multicloning cassette, respeclively, were processed in parallelas positive and negative controls. Uninduced whole cell lysates werealso examined.

[1945] Identification and Characterization SMEZ, SMEZ2, SPE-G, SPE-H andSPE-J is given in Profit T et al. J. Exp. Med. 189: 89-101 (1999).

[1946] Identification of Novel SAgs. The novel SAgs are identified bysearching the S. pyogenes M1 genome database at the University ofOklahoma with highly conserved β5 and a4 regions of streptococcal andstaphylococcal SAgs, using a TBlastN search program. The ORFs aredefined by translating the DNA sequences around the matching regions andaligning the protein sequences to known SAgs using the computer programGap. Multiple alignments and dendrograms are performed with Lineup andPileup. We used the FAST A program for searching the SwissProt (AmosBairoch, Switzerland) and PIR (Protein Identification Resource) proteindatabases. The leader sequences of SPE-G and SPE-H are predicted usingthe SP Scan program. All computer programs are part of the GeneticsComputer Group package (version 8).

[1947] Cloning of smez, smez-2, spe-g, and spe-h. 50 ng of S. pyogenesM1 (ATCC 700294) or S. pyogenes 2035 genomic DNA is used as a templateto amplify the smez DNA fragment and the smez-2 DNA fragment,respectively, by PCR using the primers smez-fw(TGGGATCCTTAGAAGTAGATAATA) and smez-rev (AAGAATTCTTAGGAGTCAATTTC) andTaq Polymerase (Promega Corp.). The primers contain a terminal tag withthe restriction enzyme recognition sequences BamHI and EcoRI,respectively. The amplified DNA fragment, encoding the mature proteinwithout the leader sequence (37) is cloned into a T-tailed pBlueScriptSKII vector (Stratagene).

[1948] Spe-g and spe-h are cloned by a similar approach, using theprimers spe-g-fw (CTGGATCCGATGAAAATTTAAAAGATT-TAA) and spe-g-rev(AAGAATTCGGGGGGAGAATAG), and spe-h-fw (TTGGATCCAATTCTTATAATACAACC) andspe-h-rev (AAAAGCTTTTAGCTGATTGACAC), respectively.

[1949] The DNA sequences of the subcloned toxin genes are confirmed bythe dideoxy chain termination method using a Licor automated DNAsequencer (model 4200). As the DNA sequences from the genomic databaseare all unedited raw data, three sub-clones of every cloning experimentareanalyzed to insure that no Taq polymerase-related mutations wereintroduced. The DNA sequence of the smez-2 gene has been annotated inEMBL/Genbank/DDBJ under accession number AF086626.

[1950] Expression and Purification of rSMEZ, rSMEZ-2, rSPE-G, andrSPE-H. Subcloned smez, smez-2, and spe-g fragments are cut frompBlueScript SKH vectors, using restriction enzymes BamHI and EcoRI(GIBCO BRL), and cloned into pGEX-2T expression vectors (PharmaciaBiotech). Due to an internal EcoRI restriction site within the spe-hgene, the pBlueScript:spe-h subclone is digested with BamHI and HindIIIand the spe-h fragment is cloned into a modified pGEX-2T vector thatcontains a HindIII 3′ cloning site.rSMEZ, rSMEZ-2, and rSPE-H areexpressed in Escherichia coli DH5a cells as glutathione-S-transferase(GST) fusion proteins. Cultures are grown at 37° C. and induced for 3-4h after adding 0.2 mM isopropyl-p-D-thiogalactopyranoside. GST-SPE-Gfusion protein is expressed in cells grown at 28° C. The GST fusionproteins are purified on glutathione (GSH) agarose and the mature toxinsare cleaved off from GST by trypsin digestion. All recombinant toxins,except rSMEZ, were further purified by two rounds of cation exchangechromatography using carboxy methyl sepharose (Pharmacia Biotech). TheGST-SMEZ fusion protein is trypsin digested on the GSH-column and theflow-through containing the SMEZ is collected.

[1951] Gel Electrophoresis. All purified recombinant toxins are testedon a 12.5% SDS-polyacrylamide gel according to Laemmli's procedure. Theisoelectric point of the recombinant toxins is determined by isoelectricfocusing on a 5.5% polyacrylamide gel using ampholine, pH 5-8 (PharmaciaBiotech). The gel is run for 90 min at 1 Watt constant power.

[1952] Toxin Proliferation Assay. Human PBLs are purified from blood ofa healthy donor by Histopaque Ficoll (Sigma Chemical Co.) fractionation.The PBLs are incubated in 96-well round-bottomed microtiter plates at10⁵ cells per well with RPMI-10 (RPMI with 10% PCS) containing varyingdilutions of recombinant toxins. The dilution series is performed in 1:5steps from a starting concentration of 10 ng/ml of toxin. After 3 d, 0.1μCï [³H]thymidine is added to each well and cells are incubated foranother 24 h. Cells are harvested and counted on a scintillationcounter. Mouse leukocytes are obtained from spleens of five differentmouse strains (SJL, B10.M, B10/J, C3H, and BALB/c). Splenocytes arewashed in DMEM-10, counted in 5% acetic acid, and incubated onmicrotiter plates at 10⁵ cells per well with DMEM-10 and toxins asdescribed for human PBLs.

[1953] TCR Vβ Analysis. Vβ enrichment analysis is performed by anchoredmultiprimer amplification. Human PBLs are incubated with 20 pg/ml ofrecombinant toxin at 10⁶ cells/ml for 3 d. A twofold volume expansion ofthe culture followed with medium containing 20 ng/ml IL-2. After another24 h, stimulated and resting cells are harvested and RNA ispreparedusing Trizol reagent (GIBCO BRL). A 500 bp β chain DNA probe is obtainedby anchored multiprimer PCR, radiolabeled, and hybridized to individualVβ5 and a Cβ DNA region dot-blotted on a Nylon membrane. The membrane isanalyzed on a Storm PhosphorImager using ImageQuant software (MolecularDynamics). Individual Vβs are expressed as a percentage of all the Vβsdetermined by hybridization to the Cβ probe.

[1954] Jurkat Cell Assay. Jurkat cells (a human T cell line) andLG-2.cells (a human B lymphoblastoid cell line, homozygous for HLA-DR1)are harvested in log phase and resuspended in RPMI-10. 100 μl of thecell suspension, containing 10⁵ Jurkat cells and 2×10⁴ LG-2 cells aremixed with 100 μl of varying dilutions of recombinant toxins on 96-wellplates. After incubating overnight at 37° C., 100-μl aliquots aretransferred onto a fresh plate and 100 μl (10⁴) of Sel cells(IL-2-dependent murine T cell line) per well are added. After incubatingfor 24 h, 0.1 μCí [³H]thymidine is added to each well and cells areincubated for another 24 h. Cells were harvested and counted on ascintillation counter. As a control, a dilution series of IL-2 isincubated with Sel cells.

[1955] Computer-aided Modelling of Protein Structures. Proteinstructures of SMEZ2, SPE-G and SPE-H are created on a Silicon Graphicscomputer using InsightII/Homology software (Biosym Technologies). TheSAgs SEA, SEB, and SPE-C are used as reference proteins to determinestructurally conserved regions (SCRs). Coordinate files for SEA (1ESF),SEB (1SEB), and SPE-C (1AN8) are downloaded from the Brookhaven ProteinDatabase. The primary amino acid sequences of the reference proteins andSMEZ-2, SPE-G, and SPE-H, respectively, are aligned, and coordinatesfrom superimposed SCRs are assigned to the model proteins. The loopregions between the SCRs are generated by random choice. MolScriptsoftware is used for displaying the computer-generated images.

[1956] Methods of isolation and characterization of SPEC is carried outby the methods of Li P L et al., J. Exp. Med. 186: 375-383 (1997)

[1957] Toxin Puification. All toxins are expressed from the pGEX vectorin Escherichia colt as glutathione S transferase (GST) fusion proteinsand purified by glutathione chromatography. Mature toxins are cleavedfrom GST by trypsin digestion and purified by two rounds of cationexchange chromatography. The first round uses carboxymethyl sepharoseand the second on a POROS HS (Perceptive Systems, Cambridge, Mass.) HPLCcolumn. All toxins are resistant to trypsin digestion except SEB whichhas a single cleavage site in the disulphide loop region. This does notaffect SEB activity.

[1958] Toxin Proliferation Assays. Human peripheral blood lymphocytesare purified by Ficoll-Hypaque and incubated for 3 d at 106 cells/ml induplicate in 96-well microtiter plates in media containing varyingdilutions of recombinant toxins. 0.1 μCi [3H]thymidine is added to eachwell, and cells were incubated a further 24 h. Plates areharvested andcounted on a scintillation counter.

[1959] TCR Vβ Analysis. These are performed using the reverse dot-blotprocedure. In brief, human peripheral blood lymphocytes are incubated at10 Vml with 1 ng/ml of recombinant toxins for 3 d. The cultures areexpanded twofold with medium containing 20 ng/ml IL-2. Cells areharvested at 4 d and RNA made by standard procedures. TCR β-chainmessenger is reverse transcribed using a set of primers specific for aconserved region in all β-chain genes. Amplification of a 500-bp Vβprobe is accomplished by an anchor primer to the 5′ end of the β-chainprimers plus a single Cβ region primer. This probe is radiolabeled andreverse blotted to filters containing individual β-chain genes. Relativechanges in individual β-chain mRNA are compared to unactivated PBL.

[1960] Anti-TCR mAb FACS Staining.Activated T cells are incubated for 1h on ice with 25 ml of anti-TCR BV2 (MPB2/Cll; a gift from A. W.Boylston, Universtiy of Leeds, Leeds, UK), anti-BV551 (LC4; a gift fromR. Levy, Stanford University Medical School, Stanford, Calif.),anti-BV5S3 (42/1C1; a gift from A. W. Boylston, Universtiy of Leeds,Leeds, UK), anti-BV8.1 (C305; a gift from A. Weiss, University ofCalifornia, San Francisco, Calif.), and anti-BV12S (S511; a gift from D.Posnett, Cornell University Medical College, NY). Washed cells are thenincubated with 1 ml FITC goat anti-mouse (Becton Dickinson) andincubated on ice for a further 30 min. After washing, cells are analyzedon a FACSCAN®.

[1961] Zinc Blots. Recombinant toxins (10 (ug) are incubated intriplicate with 10 μM EDTA followed by 100 μM 65ZnCl (New EnglandNuclear, Boston, Mass.) in 20 mM Tris, pH 8.0, 10 mM MgCl2, 0.15 M NaClmade zinc free by addition of chelex resin (Sigma Chemical Co.). Samplesare then dot blotted to nitrocellulose filters using a 96-well dot-blotapparatus. Filters are washed briefly three times with zinc-free buffer,and then autoradiographed. Spots are cut out and counted on gammacounter (Packard Instrs., Meriden Conn.) to quantify 65Zn bound to eachtoxin.

[1962] Nondenaturing SDS Electrophoresis. Toxin samples are incubated instandard Laemmli reducing sample buffer (containing 1% SDS and 10 mMdithiothreitol), and then resolved as normal of a 12.5% acrylamide gel.For denaturing conditions, samples are heated,to 100° C. for 2 minbefore loading. To prevent dimers from dissociating during running, thepower is. maintained below 2 W (20 mA and 100 V). Some reduced samplesare treated with 20 mM iodoacetic acid, pH 7.0, before loading. EDTA isadded to some samples at 10 mM. incubation at 37° C. as the percentageof LG-2 cells in aggregates, and is determined by light microscopy.

[1963] Western Blotting of S. pyogenes Strain 2035-derived SPE-C. S.pyogenes strain 2035 is grown under anaerobic conditions in brain heartfusion medium at 37° C. for 24 h without shaking. Supernatant proteinsare concentrated by sequential (NH₄)₂SO₄ precipitations, with cuts of<40%, 40-60% 60-80%, and 80% saturation, and resuspended in 50 mM Tris-1mM EDTA, pH 7:4, at 500-1,000 times their original concentration.Recombinant SPE-C or 10 μl of the 60-80% (NH₄)₂SO₄-precipitable fractionof 2035 supernatant are combined with an equal volume of nonreducing 2%SDS sample buffer and separated by 0.1% SDS-12% PAGE. Denatured samplesare heated (95° C.) for 2 min before analysis. Fractions are dialyzedextensively against 25 mM Tris-50 mM NaCl-1 mM EDTA, pH 7.4, to removesalt. After separation by SDS-PAGE, proteins are transferred to anitrocellulose filter (Hybond-C; Amersham Corp., Arlington Heights,Ill.) in an electroblotting apparatus using Towbin buffer lacking SDS(25 mM Tris.HCl, pH 8.5-150 mM glycine-10% methanol). The filter isblocked in PBS-0.05% Tween-5% nonfat dried milk powder-0.1%) normalrabbit serum and stained with 1:6,000 peroxidase-labeledaffinity-purified rabbit and-SPE-C immunoglobulin. The peroxidaseconjugate is detected on radiographic film by chemiluminescence (ECL;Amersham Corp.) according to the manufacturer's instructions.

[1964] Size Exclusion Chromatography. Recombinant SPE-C. (2 mg/ml) isdialyzed at 4° C. overnight in 20 mM BisTris-Tris, pH 6.0 or 9.0. 20 μïsamples are diluted into 100 μï 50 mM BisTris, pH 6.0, 7.0, 8.0, or9.0/0.1 M NaCl and incubated for 1 h at room temperature beforeseparation at 1 ml/min (±0.05 ml/min) on a Superosel2 (Pharmacia) highresolution HPLC column attached to a Biocad Sprint (Perceptive Systems)preequilibrated with the respective incubation buffer. Tracechromatograms monitoring Abs_(280nm), pH, and conductivity are allrecorded directly and subsequently analyzed for retention times, peakintegration, and peak assignment using the on-line Biocad software.Traces are grouped and printed using the stacked trace mode whichautomatically aligns each trace to the injection point.

[1965] Identification and Characterization of the Staphylococcalenterotoxins SEG, SEH, SEI, SEJ, SEK, SEL, SEM is carried out by themethod of Jarraud S J Clinical Microbiology (1999).

[1966] Strains. S MJB1316 (a gift from Sibyl Munson, University ofWisconsin, Madison, Wis.), an RN450 derivative that contains the clonedseg gene on the staphylococcal expression vector pRN5548, is used as segpositive control. The following S. aureus strains were used to check thespecificity of PCR amplification: FDA-S6 (ATCC 13566 (sea+ seb+)),FRI-137 (ATCC 19095 (sec+, seg+, seh+, sei+)), FRI-1151 m (sed+),FRI-326 (ATCC 27664 (see+)), FRI-569 (ATCC 51811 (seh+)), FRI-1169(tst+), TC-7 (eta+, seg+, sei+), and TC-146 (etb+seg+ sei+). Two hundredthirty S. aureus clinical isolates are collected. They are isolated from58 patients with S. aureus infection (arthritis, skin infection,pneumonia, or infective endocarditis), 102 patients with acute toxemia(TSS, SSF, or SSSS), and 70 asymptomatic nasal carriers. All strains arecollected from hospitals located throughout France and are identified asS. aureus by their ability to coagulate citrated rabbit plasma(bioMerieux, Marcy-I'Etoile, France) and to produce a clumping factor(Staphyslide Test; bioMerieux). Escherichia coli TG1 is used for plasmidamplification and genetic manipulations.

[1967] DNA amplification and sequencing DNA is extracted from A900322cultures and used as a template for amplification with primers sei-1 andseg-2. Primers wsei and wseg are designed following identification ofsuitable hybridization sites in the sei and seg genes and werecompatible with the Clontech Genome Walker kit (Ozyme; Montigny-LeBretonneux, France), which is suitable for cloning unknown DNA sequencesadjacent to a known sequence. This kit is used, according to thesupplier's instructions, to identify sei and seg flanking regions usingprimers hindIII and wsei on a HindIII chromosomal digest for theamplification of the sei-upstream region; and primers hpaI and wseg onan HpaI chromosomal digest for the amplification of the seg-downstreamregion. PCR products are analyzed by electrophoresis through 0.8%agarose gels (Sigma, St. Louis, Mo.), purified using the High Pure PCRProduct Purification kit (Boehringer Mannheim, Meylan, France), andsequenced (Genome Express, Grenoble, France). Sequences are compiled,analyzed, and compared using Blast (http://www.ncbi.nlm.nih.gov/BLAST),GeneJokey, and ClustalX software (European Bioinformatics Institute,Cambridge, U.K., http://www.ebi.ac.uk).

[1968] Toxin-gene detection. Sequences specific for sea-e, seg-i, tst,eta, and etb, encoding SEA-E, SEG-I, TSST-1, ETA, and ETB, respectively,are detected by PCR. DNA from clinical isolates is extracted fromcultures and used as a template for amplification with the primersdescribed in Table 1 (Eurogentec, Seraing, Belgium). Table 1 of primersused is given-in Jarraud et al., J. Clin. Micro. (1999). Amplificationof gyrA is used as a control to confirm the quality of each DNA extractand the absence of PCR inhibitors. All PCR products are analyzed byetectrophoresis through 1% agarose gels (Sigma).

[1969] Detection of bacterial RNA by RT-PCR. Total RNA is extracted fromstaphylococcal cultures by using RNeasy spin columns (Qiagen,Courtaboeuf, France). cDNA is synthesized using Ready-To-Go RT-PCR beads(Pharmacia Biotech, Orsay, France) by incubating 0.1 μg of total RNAwith the following pairs of primers (primer 5′, sel3), (sel-4, sel-5),(sel 1, sel2), (invsel2, invsem1), (sem1, invsei1), (sei1, sei2),(invsei2, ψent2), (ψent1, invsek 1), (sek1, sek2), (invsek2, invseg1),(seg1, seg2), (invseg2, primer 3′). The reaction mixtures are incubatedwith each primer pair described above, at 42° C. for 30 min for reversetranscription, followed by 30 cycles of amplification (1-mindenaturation at 94° C. 1-min annealing at 55° C., and 1-min extensionat72° C.). The RT-PCR products are then analyzed by electrophoresisthrough 1% agarose gel. RNA extracts are tested for DNA contamination bypreincubating the reaction mixtures at 95° C. for 10 min to inactivatereverse transcriptase before the RT-PCR.

[1970] Production and purification of recombinant enterotoxins. Primersare designed following identification of suitable hybridization sites insel, sem, sei, sek, and seg. The 5′ primers are chosen within the codingsequence of each gene, omitting the region predicted to encode thesignal peptide, as determined by hydrophobicity analysis according toKyte and Doolitttle with GeneJockey software and SignalP V1.1 World WideWeb Prediction Server (www.cbs.dtu.dk/services/SignalP/); the 3′ primersare chosen to overlap the stop codon of each gene. A restriction site isincluded in each primer. DNA is extracted from A900322 or MJB1316 andused as a template for PCR amplification. PCR products and plasmid DNAare prepared using the Qiagen plasmid kit. PCR fragments were digestedwith EcoRI and PstI (Boehringer Mannheim) and ligated (T4 DNA ligase;Boehringer Mannheim) with the pMAL-c2 expression vector from New EnglandBiolabs (Ozyme) digested with the same restriction enzymes. Theresulting plasmids are transformed into E. coli TG 1. The integrity ofthe ORF of each construct is verified by DNA sequencing of the junctionbetween pMAL-c2 and the different inserts. The fusion proteins arepurified from cell lysates of transfected E. coli by affinitychromatography on an amylose column according to the supplier'sinstructions (New England Biolabs).

[1971] T cell proliferation assays. PBL from healthy donors are culturedin 24-well plates (10⁶ cells/well) in RPM11640 medium supplemented with8% pooled human serum and 10 μg/ml recombinant staphylococcal toxin.rIL-2 (50 IU/ml) is added on day 5. When necessary, T cell cultures arediluted in IL-2-supple-mented medium until TCR analysis. For controls Tcells from the same donors that are stimulated with 0.5 μg/ml Phaseolusvulgaris leucoagglutimn (PHAL) (Sigma) are used.

[1972] Flow cytometry. The following mAb (mAb; specificity indicated inbrackets) are used for flow cytometry: E2.2E7.2 (Vβ2), LE89 (Vβ3),IMMU157 (Vβ5.1), 3D11 (Vβ5.3), CRI304.3 (Vβ6.2), 3G5D15 (Vβ7), 56C5.2(Vβ8.1/8.2), FIN9 (Vβ9), C21 (Vβï 1), S511 (Vβ12), IMMU1222 (Vβ13.1)JIJ74 (Vβ13.6), CAS1.1.13 (Vβ14), Tamaya1.2 (Vβ16), E17.5F3 (Vβ17),BA62.6 (Vβ18), ELL1.4 (Vβ20), IG125 (Vβ21.3), IMMU546 (Vβ22), andHUT78.1 (Vβ23). These mAb, and CD4- and CD5-specific mAb, is purchasedfrom Beckman/Couker/Immunotech (Marseille, France). Cells are phenotypedby indirect immunofluorescence, as described previously. Briefly, cellsare incubated with unconjugated mAb for 30 mm at room temperature, thenwashed and incubated with FITC-conjugated rabbit anti-mouse Ig antiserum(BioAtlantic, Nantes, France) for 30 min on ice. After washing, cellsare analyzed by flow cytometry on a FACScan apparatus (Becton Dickinson,Mountain View, Calif.) using the LYSYS II software package on aFACstation.

[1973] Immunoscope analysis. Total RNA is extracted using the Tnzolreagent (Life Technologies, Gaithersburg, Md.). TCR β-chain-specificprimers are as described previously, and reverse transcription, PCRamplification, and run-off steps are performed as reported previously.Fluorescent DNA products are loaded on a sequencing gel and analyzedwith the Immunoscope software.

[1974] Identification of the seg and sei flanking regions. When thiswork was initiated, the coding regions of only seg and sei wereavailable, and the two genes were known to be in tandem orientation,separated by a 1.9-kb DNA fragment in S. aureus strain A900322. A 3.2-kbfragment is thus amplified by PCR with primers sei1 and seg2 and wasthen sequenced. The intergenic 1.9-kb DNA sequence contains three openreading frames (ORF1, 2, and 3) of 399, 327, and 777 bp, respectively.Comparison of the deduced amino acid sequences of these ORFs withtranslated sequences from GenBank showed that the putative proteinscorresponding to these ORFs had substantial sequence similarities toknown SEs: ORF1 exhibited homology to the N-terminal region of SEB; ORF2to the C-terminal region of SEC; and ORF3 to SEA. The PCR “walking”strategy is chosen to identify the seg and sei flanking regions. The useof primers wsei and hindIII on HindIII digests amplifies andallowssequencing of the 3.2 kb of DNA upstream of sei. Analysis of thissequence showed two significant ORFs (ORF4 and ORF5) of 783 and 720 bp,respectively. ORF4 exhibited homology with SEJ, and ORF5 with SEI. Theuse of primers wseg and hpaI on HpaI digests amplified a 0.8-kb fragmentdownstream of seg. Sequence analysis of this fragment reveals no othersignificant ORFs. The concatenated sequence of seg-sei-intergenic,-upstream and -downstream regions is validated by sequencing a 6.189-kbPCR fragment encompassing the whole region. Although sei in strainA900322 is 100% homologous with the sequence deposited in GenBank(accession number AF064774), seg in strain A900322 showed one mutation,corresponding to a Leu→Pro substitution at position 29. This new variantis designated SEGL29P. ORFs 1-5 arehomologous but not identical with anyknown enterotoxins hence they corresponded to new enterotoxins. However,ORF1 and 2 are at least 50% shorter than any of the known enterotoxins.ORF-1 possesses a satisfactory Shine-Dalgarno (SD) sequence(TGGAGT-N7-AUG, consensus AGGAGG-N6/10-AUG) but, in comparison with SEB,to which it is highly related, shows a large deletion of its 3′ end,which corresponds to a region that is essential for biological(superantigenic) activity. ORF2 has neither an SD sequence nor a signalpeptide, and resembles an N-terminal-truncated SEC. Accordingly, ORF1and 2 are designated ψ ent1 and 2, respectively, meaning they representpseudogenes with no likely biological function. In contrast, ORFs 3, 4,and 5 have sizes consistent with active enterotoxin-like molecules. ORF5possesses a satisfactory SD sequence and translation start site, whereasORF3 and ORF4 have an adequate SD sequence in front of a noncanonical,although suitable, translation start site (ATT) coding the thionine.Thus. ORF3, ORF4, and ORFS are designated sek, sel, and sem,respectively. Thus, the 6301-bp DNA region identified contains seg andset plus three potential enterotoxin genes (sek, sel, and sem) and twopseudogenes (ψ ent1, ψ ent2), all in the same orientation. We designatedthis region egc for “enterotoxin gene cluster.” With the exception ofplasmid pIB485, which contains sed and sej in opposite orientationsseparated by 895 nucleotides, and the staphylococcal pathogenicityisland, which contains tst and ent separated by 10.234 kb, no such genecluster organization has been previously described for enterotoxingenes.

[1975] Transcriptional analysis. To investigate whether this segtranscript was polycistronic, i.e., encoded one or more of the ORFsidentified in egc, c-DNA is generated from strain A900322 total RNA byreverse transcription and amplified by PCR using primer pairs locatedwithin each gene and bracketing adjacent genes. Abundant RT-PCR products(B to K) of the expected size are obtained using the correspondingprimer pairs. In contrast, no RT-PCR product A (primer 5′, seI3) nor L(primer invseg2 and primer 3′) is obtained. These results suggest thatthe seven genes and pseudogenes composing egc are cotranscribed, andthat the 5′ and 3′ ends of the transcript must be close to the beginningof sel and to the end of seg, respectively. Sequence analysis revealputative −10 and −35 promoter sequences (TTGTCT-N15-TAATTT-N134-ATT)upstream of the sel start codon. The 3′ end may lie at an invertedrepeat at position 6018-6067, which is a potential transcriptiontermination signal, 5830 nucleotides downstream of the putativetranscription start site. These results suggest that egc is an operon.

[1976] Superantigen activity. The association of related genes that arecotranscribed suggested that the resulting peptides might havecomplementary effects on the host's immune response. One hypothesis isthat gene recombination created new variants of toxins differing bytheir superantigen profiles. Purified recombinant SEL, SEM, SEI, SEK,and SEGL29P expressed in E. coli are studied for their ability to induceselective expansion of T cells bearing particular TCR Vβ regions inshort-term PBL culture. As shown in Tables III and IV, recombinant SELSEM, SEI, and SEK consistently induces selective expansion of distinctsets of Vβ subpopulations. By contrast. SEGL29P fails to triggerexpansion of any of the 23 Vβ subsets. The sum of results obtained witheach of these recombinant toxins globally corresponds to the selectiveexpansion of Vβ subpopulations induced by crude supernatant ofstaphylococcal culture of strains that harbored egc (data not shown).This suggests that the maltose-binding protein portion of the fusiontoxins do not significantly influence the Vβ specificity of thesesuperantigens. To investigate whether the L29P mutation could explainthe lack of superantigen activity, a rSEG with an L29 codon isconstructed from S. aureus strain MJB1316 (which contains the cloned segon a plasmid) and then expressed in E. coli, and the superantigenactivity of this toxin is tested. SEGL29.induces selective expansion ofVβ14 and, to a lesser extent, Vβ13.6,OT cells (Table III). The L29Pmutation thus accounts for the complete loss of superantigen activity.Computer modeling of the two-dimensional structure of the wild-type andmutated proteins reveals no major conformational differences between thetwo proteins (not shown). It is likely that L29 is located at a positioncrucial for proper superantigen/MHC II interaction. In addition to theselective expansion of TCR Vβ subsets observed with the differenttoxins, flow cytometry reveals preferential expansion of CD4 T cells inSEI and SEM cultures. By contrast, the CD4/CD8 ratios in SEK-, SEL-, andSEG-stimulated T cell lines are close to those in fresh PBL. Thisphenomenon, which is observed with cells from several donors, reflects avariable contribution of the CD4 coreceptor to the T cell activationprocess, depending on the affinity of the TCR for the superantigen/MHCcomplex. To document the TCR Vβ composition of superantigen-stimulated Tcell lines and the clonal diversity of the expanded TCR Vβ subsets, thesize distribution of PCR-amplified TCR β-chain junctional products isstudied using the Immunoscope technique. Results of this molecularanalysis are in good overall agreement with those obtained by flowcytometry, as similar dominant TCR Vβ subsets are identified with thetwo approaches. Additionally, Immunoscope analysis shows that thecomplementarity-determining region 3 size distribution of TCR β-chainjunctional transcripts within expanded Vβ subsets is pseudogaussian inall superantigen-stimulated cultures, reflecting a high level ofpolyclonality. This is further confirmed by sequence analysis of TCR βjunctional transcripts derived from some expanded TCR Vβ subsets. Takentogether, these TCR repertoire studies confirm the superantigenic natureof the new toxins identified in this study.

EXAMPLE 63 The Identification and Characterization of the SETs

[1977] See, Williams et al., Infect. Immun. 68: 4407-4414 (2000). Afragment of the set1 gene is initially cloned using a screening methodincorporating alkaline phosphatase (PhoA) as a reporter molecule toidentify secreted proteins, as previously described for Streptococcuspneumomae. This system utilizes an alkaline phosphatase-negative strainof Escherichia coli CC118 (PhoA−) and plasmid pHRM104, which contains atruncated E. coli phoA gene lacking its signal sequence. Thus, for thesecretion of functional alkaline phosphatase activity, genes encodingsignal sequences must be cloned in frame with the phoA gene in thereporter system. A 5 ug sample of S. aureus NCTC 6571 genomic DNA isdigested with Sau3A, fragments of 1 to 4 kb were ligated to pHRM104which is linearized with BamHI and the ligations are used to transformE. coli CC118. Transformants exhibiting alkaline phosphatase activityare detected by growth on nutrient agar containing5-bromo-4-chloro-3-indolylphosphate. Plasmid DNA is prepared from allpositive clones, and the sequences of the inserts are determined. One ofthese clones, pExp5, contains a 237-bp DNA fragment at set1.

[1978] Identification of set: An alkaline phosphatase fusion strategy isemployed to identify proteins exported by S. aureus. Using this system,one clone is isolated, pExpS, that contains DNA encoding the N-terminal79 amino acids of a secreted protein fused to phaA. Comparison of thenucleotide sequence of pExpS with those in the Oklahoma Universitysequencing project of S. aureus NCTC 8325(http://www.genome.ou.edu/staph.html) revealshomology (91%) to contig386 (designation as of July 1998). Contig 386 contains a partial openreading frame encoding a putative 191-amino-acid protein (ORF386), and asearch of the SwissProt database reveals that ORF386 has approximately25% identity to a number of staphylococcal and streptococcal exotoxins.The homology includes two regions that are similar to the consensusstaphylococcal enterotoxin/streptococcal exotoxin signatures (PROSITEand PCOC00250) (12, 16). The first of these signatures is awell-conserved region found in the majority of the staphylococcalenterotoxins but not TSST-1, while the second signature is a morediffuse sequence which is present in both the enterotoxins and TSST-1.This similarity suggested that ORF386 codes for a novel exotoxin. TheDNA sequence identified in pExpS as encoding an N-terminal fragment(including the signal peptide sequence) of an unknown protein and thatof ORF386 are very similar although not identical. The gene within pExpSalso encodes a novel exotoxin-like. protein. This open reading frame isdesignated set1 (for staphylococcal exotoxin-like gene 1).

[1979] Cloning of the full-length set1 gene and identification of thenovel set gene cluster: A set1-specific probe (253 bp) is PCR amplifiedusing pExp5 as template DNA and the primer pair5′-GGGACAGAATAATACTATGAAATTAA AAACG and 5′-ATCTTTTTGGTTAAAGCGTAC.Labeling of the set1 probe DNA with digoxigenin-dUTP (DIG-dUTP),hybridization, and subsequent immunological detection are carried out asspecified by the manufacturer (Roche Diagnostics Ltd.). Southern blotsof genomic DNA digested with an array of restriction endonuclcascs andprobed with the set1 probe reveals that this gene is present on a 5.2-kbEcoRI fragment. Chromosomal DNA is digested with EcoRI and ligated topUC19. The ligation mix is transformed into E. coli TOP10. Transformantsare transferred to Hybond-N membranes (Amersham, Little Chalfont, UnitedKingdom) and screened for the sel1 gene using the 253-bp set1 probe. Oneclone, pSETlfi, has a 5.2-kb insert containing the set1 gene. The 5.2-kbinsert is sequenced by primer walking using a BigDyc terminator kit asspecified; by the manufacturer (ABI Perkin-Elmer). The cycle-sequencingreactions are run on an ABI 310 genetic analyzer. The template issequenced in both directions in duplicate.

[1980] Cloning of the full-length set1 gene and identification of thenovel exotoxin-like gene cluster. The set1 gene is mapped to a 5.2-kb£coRI S. aureus genomic DNA fragment by using a 253-bp probe designed totarget the 5′ end of set1 (data not shown). To clone this fragment, acomplete S. aureus NCTC 6571 genomic DNA-pUC19 EcoRI library isconstructed in E. coli and screened using the 253-bp set1 probe. Onepositive clone, pSET16, containing a 5,175-bp fragment is used forfurther analysis. The complete DNA sequence of this 5,175-bp fragment isdetermined and has been deposited in GenBank (accession no. AF094826).Analysis of the DNA sequence of pSET16 reveal four complete open readingframes together with two partial open reading frames at the 5′ and 3′extremities of the DNA, A BLAST search of the SwissProt databaseindicated that the four complete open reading frames and one of thepartial open reading frames (5′ end) had homology, although low, tothose encoding a number of staphylococcal and streptococcal exotoxins,including TSST-1. The second partial open reading frame at the 3′extremity encoded the start of a possible host specificity determinantmethylase (HsdM)-like protein (54% identity over 26 amino acids toKlebsiella pneumoniae HsdM). The novel exotoxin-like genes aredesignated set1 to set5 based on the order of their discovery. Theorganization of the set genes is shown in Williams et al., supra. Thedistance between each of the set genes varies from 339 to 446 bp, whilethe shorter intergenic region between set4 and the isdM-like gene is 261bp. Apart from the set2 gene (for which the upstream region has not beensequenced), all genes are preceded by putative ribosome-binding sites 7bp upstream of their start codons.

[1981] Computer-aided modeling of protein structures: The SET sequences,excluding SET2, for which we did not have complete sequence data, weremodeled against two target proteins, TSST-1 and SPEC, using a SiliconGraphics computer. Coordinate files for SPEC (1 an 8) and TSST-1 (2tss)are downloaded from the Brookhaven Protein Database. MODELLER softwareis used which optimizes spatial restraints derived from the sequencealignments, to build models of the SET proteins. Protein models thusderived are viewed using MOLSCRIPT. Based on sequence alignments usingCLUSTAL_X Windows 1, the SET3 and SET4 sequences are modeled usingTSST-1 (2tss.pdb) as template while the SET1 and SETS sequences aremodeled using SPEC (1an8.pdb).

[1982] Distribution of the set1 gene among staphylococci.:Staphylococcal strains are screened for set1 by PCR. Chromosomal DNAfrom S. aureus NCTC 6571, NCTC 8325-4, FRI326, and eight clinicalisolates (obtained from Geoffrey Scott, University College LondonHospital) and S. epidermidis NCTC 11964 and NCTC 11047 are used astemplates. A total of 30 cycles of amplification are performed using theforward primer 5′-GAATTCAGATTGGGAGAATAATACT ATG and the reverse primer5′-AGATCTCAACGTTTCATCGTTAAGCTGC.

[1983] Distribution of the set1 gene among staphylococci.: Ten strainsof S. aureus, FRI326, NCTC 8325-4, and eight clinical isolates, togetherwith two strains of S. epidermidis (NCTC 11964 and NCTC 11047) areanalyzed by PCR for the presence of the sel1 gene. Products aregenerated for all of the S. aureus strains but not for either of the S.epidermidis strains (Williams et al., supra). Cloning and sequencing ofthe sel1 genes from three of the S. aureus strains (FRI326, NCTC 6571,and NCTC 8325-4) reveals that the NCTC 6571 gene has 92 and 88% identityto the FR1326 and NCTC 8325-4 sel1 genes, respectively. The respectiveGenBank accession numbers for the set1 genes from FRI326, NCTC 8325-4,and NCTC 6571 are AF188836, AF188837, and AF188835. The predictedprotein sequences differ in 30 (FRI326) and 34 (NCTC 8325-4) positionsfrom the NCTC 6571 SET1 sequence, corresponding to 87 and 84% identity,respectively, at the amino acid level.

[1984] Dot blot analysis of set1 mRNA production.: Overnight cultures ofS. aureus grown in 1% casein hydrolysate-2.5% yeast extract are diluted(1:50) in fresh broth and incubated at 37° C. with aeration. Aliquotsare taken at various time points, and the cells are harvested and washedwith 0.5% Twccn 80. The cell pellets are suspended in 0.5 ml of lysissolution (consisting of 9.6 ml of Divolab no. 1 detergent [DiversyLeverLtd.], 24 ml of 500 mM sodium acetate [pH 4], 66.4 ml of distilledwater), 0.5 ml of acid-phenol (pH 4), and 0.1 ml of chloroform-isoamylalcohol (24:1) and lysed using a Ribolyser (Hybaid). After a 10-minincubation on ice followed by ccntrifugation, the supernatants areextracted with phenol-chloroform and the RNA is precipitated. RNA (2 μg)is blotted onto a nylon membrane and probed using a DIG-labeled set1probe. The hybridization and subsequent chemiluminescent detection arecarried out as specified by the manufacturer (Roche Diagnostics Ltd.).

[1985] Dot blot analysis of set1 mRNA production.: To determine if set1is expressed in S. aureus, total cellular RNA extracted at variousgrowth stages from S. aureus NCTC 6571 is probed with a probe specificto set1. The expression of set1 is growth phase dependent, with thehighest level of expression in the stationary phase.

[1986] Cloning and expression of set1.: The set1 gene encoding themature protein minus the signal peptide is PCR amplified from pSET16 DNAand inserted into the pQE30 N-terminal histidine tag fusion vector. Thisconstruct is introduced into E. coli JM109. Protein production is foundto be most efficient when bacteria are grown at 28° C. after induction(data not shown), giving yields of approximately 20 mg of solubleprotein per liter. Analysis of SET1 by gel filtration chromatog-raphyreveals that it exists as a monomeric protein of about 23 to 24 kDa. Theset1 genes from NCTC 8325-4 and FRD26 arealso cloned and expressed usingthis system and found to encode monomeric proteins with molecular massesidentical to that of SET1 from NCTC 6571. All rSET proteins arehomogenous as determined by sodium dodecyl sulfate-polyacryl-amide gelelectrophoresis and migrate with molecular masses of about 23 to 24 kDa.

[1987] Cloning of set1 into an N-terminal polyhistidine expressionvector.: The oligonuclcotide 5′-GGATCCGCAGAAAAACAAGAGAGAGTAC and5′-GTCGACAACGTTTCAf CG-TTAAGCTGCC are designed to amplify the 677-bpset1 gene minus the DNA encoding the N-terminal signal peptide and alsocontained recognition sequences for the restriction enzymes BamHI andSalI (underlined), respectively. The PCR fragment is initially clonedinto PCR2.1-TOPO and transformed into TOP10 (Invitrogen, Leek, TheNetherlands). The set1 gene is extracted from PCR2.1-TOPO on aBamHI-SalI fragment and ligated to BamHI-SalI digested pQE30 (QiagenLtd., Crawley, United Kingdom). The ligation mix is transformed into E.coli JM109(pREP4), and transformants are selected by growth at 30° C. onLuria-Bertani agar containing 100 μg of ampicillin per ml and 25 μg ofkanamycin per ml.

[1988] Expression at set1 and purification of recombinant SET1.: Forgene expression, positive clones aregrown overnight in Terrific broth,diluted 1:25 in fresh broth, and incubated for a further 1 h at 37° C.Gene expression is induced with 1 mM isopropyl-p-D-thiogalactopyranosidc(IPTG) for 4 h at 28° C. Cells are harvested by centrifugation at6,000×g for 30 min and then resuspended and lysed for 20 min in B-PERprotein extraction reagent (Pierce & Warriner Ltd.) containing 20 mMimidazole, 1.5 μM phenylmethylsulfonyl fluoride, 1 μM pepstatin, and 10μM leupeptin. Lysates are clarified by centrifugation at 23,000×g for 15min. Recombinant proteins are purified using Ni-nitrilotriaceticacid-agarose columns under native conditions as specified by themanufacturer (Qiagen Ltd.), except that after the lysates are loadedonto the column, an additional column wash, consisting of 2.5 mg ofpolymyjtin B per ml in wash buffer, is performed to remove contaminationwith lipopolysaccharide. Finally, recombinant SET1 (rSET1) is furtherpurified by gel filtration chromatography using a Superdex75 columnprecquilibrated in phosphate-buffered saline and attached to a PharmaciaSMART system (Amersham-Pharmacia Biotech).

[1989] Assay of cytokine production by PBMCs.: PBMCs are prepared aspreviously described. Cells are plated at a density of 2 X 106 cells/mlin 24-well plates and stimulated with graded concentrations of protein.SEB (Sigma-Aldrich Ltd.) is used as a positive control. Cellsupernatants are assayed for the presence of interleukin-1β (IL-1β),IL-6, and tumor necrosis factor alpha (TNF-α) using two-siteenzyme-linked immunosorbent assays as previously described.

[1990] Capacity of rSET1 to stimulate production of the proinflammatorycytokines IL-1β, IL-6, and TNFα: All characterized staphylococcalenterotoxins are able to stimulate proinflammatory cytokines. Theability of rSET1 from strain NCTC 6571 to stimulate the production ofthe cytokines IL-1β, IL-6 and TNF-α by PBMCs is compared to that of SEE.To establish the purity and to ensure that similar concentrations ofeach protein preparation are used, rSET1 and commercial SEE are comparedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.Stimulation of PBMCs with rSET1 results in the production of all threeof the proinflammatory cytokines tested (Williams et al., supra). rSET1stimulates the secretion of IL-6 by PBMCs over the dose range of 100ng/ml to 10 μg/ml, compared to SEB, which is active at inducing IL-6production over the dose range of 10 ng/ml to 10 μg/ml. However, themaximal levels of IL-6 produced in response to rSET1 are almost 10-foldgreater than those induced by SEB. Heating of rSET1 at 100° C. for 20min reduces its ability to stimulate IL-6 production by 69%. SEB is avery poor inducer of IL-1β secretion by PBMCs. rSET1, on the other hand,shows a similar dose response to that produced for IL-6 secretion, beingactive over the range of 100 ng/ml to 10 μg/ml. The maximal level ofIL-1β produced by PBMCs stimulated with rSET1 is, again, almost 10-foldgreater than that induced by SEB. rSET1 is not a potent inducer of TNF-αsecretion, being active only over the concentration range from 1 to 10μg/ml. In contrast, SEB was a potent inducer of TNF-α secretion byPBMCs, having activity over the dose range of 1 ng/ml to 10 μg/ml. Onceagain, the maximum level of cytokine secretion is produced in responseto rSET1 and not to SEB; however, the levels are only slightly higher.

[1991] The nuclcotide sequence data reported here have been deposited inthe GenBank database under accession numbers AF094826 (set gene dusterfragment), AF188835 (NCTC 6571 set1 gene), AF188836 (FRI326 set1gene),and AF188837 (NCTC 8325-4 set1 gene).

EXAMPLE 64 Isolation and Characterization of SPEB

[1992] SPEB by Purification of native SpeB from strain MGAS 1719 isgiven by Gubba S et al., Infect. Immun. 66: 765-770 (1998). Native28-kDa SpeB protease was purified from the culture supernatant of strainMGAS 1719 as described previously (14). The purified protein is >95%pure, as analyzed by SDS-PAGE and staining with Coomassie brilliantblue. Amino-temtinal sequencing of recombinant proteins. A 20 pg aliquotof each recombinant protein with the His tag removed by digestion withrTEV protease is analyzed by SDS-PAGE, transferred to a Problottmembrane (Applied Biosystems, Foster City, Calif.), and stained withCoomassie brilliant blue. The desired protein band is excised andanalyzed with an Applied Biosystems model 473A protein sequencer locatedat the Baylor College of Medicine Core Protein Facility.

[1993] Construction of the speB C192S mutant is given by Musser J et al,Infect. Immun. 64: 1913-1917 (1996). The speB gene is amplified from S.pyogenes MGAS 1719, a strain that contains the speB7 allele (24), andthe PCR fragment is doned into the pCR-Script vector (Stratagene, LaJolla, Calif.). The oligonucleotides used in PCR amplificationcorresponds to positions −160 to −138 (primer SPEBX) and 1303 to 1325(primer SPEB2) of a published yeB gene sequence. The 30-ul PCR mixturecontained 1 μg of MGAS 1719 genomic DNA, 50 mM KC1, 10 mM Tris-HCl (pH8.5), 1 mM Mg²⁺, 200 μM each deoxynudeoside triphosphate, 2 μM eachprimer, and 2.5 U of Pfu DNA polymerase. Amplification is performed in aPerkin-Elmer Cetus model 480 thermocyder, with the following parameters:30 cycles of denaturation at 94° C. for 1 min, annealing at 55° C. for 1min, and extension at 72° C. for 1.5 min. A 1.5-kb amplificationfragment is cloned into pCR-Script, and plasmid DNAs obtained fromEscherichia coli transformants are characterized by automated DNAsequencing (24) to verify that no mutations occurred in the insertedspeB gene.

[1994] One clone (pSEB1719) with the same nudeotide sequence frompositions −155 to 1325 as the speB7 allele is chosen for use inmutagenesis. A PCR-based mutagenesis procedure is used to generate aC-192 (TGT)-to-S-192 (AGT) substitution. The sense-strand primer (TGT2)has the sequence CAG GAAGTGTTGCTACTGCAA, and the sequence of theantisense-strand primer (TGT1) is CAACACTTCCTGTAGCTGCATG. Separate PCRsare done with primers TGT2 and SPEB2 in one reaction and primers TGT1and SPEBX in a second reaction. These reactions are carried out with 1ng of plasmid pSEB1719 and the PCR conditions described above exceptthat primer annealing is done at 40° C. for 1 min. As expected, two DNAfragments (740 and 757 bp) are generated by the reactions. Followingagarose gel electrophoresis, these fragments are recovered and pooled,and 1 μg of the mixture is used in a second round of PCR amplificationwith primers SPEBX and SPEB2 and an annealing temperature of 55° C. Aresulting 1.5-kb full-length speb gene is then cloned into pCR-Script.Screening of plasmids from 183 colonies by agarose gel electrophoresisidentified five organisms with appropriately sized inserts. The targetregion was sequenced in these five clones, and one (pSEBC2S) that hasthe desired TGT->AGT (C192S) substitution is identified. The 1.5-kbinsert is then sequenced in its entirety by an automated strategy (24),and no additional nudeotide substitutions are identified.

[1995] Expression and purification of recombinant proteins is given byGubba et al. supra 1998 Recombinant proteins are obtained from E. coliBL21 containing the appropriate plasmids after induction withisopropyl-p-o-thiogalactopyranoside (IPTC). Cells are grown overnight at30° C., diluted 1:10 in Luria-Bertani medium supplemented with 100 μg ofampicillin per ml, and cultured to an optical density at 590 nm of 0.5to 1.0. IPTG is added (final concentration, 0.6 mM), and the cells aregrown for an additional 2 to 6 h. After centrifugation at 10,000×g for10 min, the supernatant is discarded and the cell pellet is stored at−70° C. The cells are resus-pended in 5 volumes of 50 mM Tris-HCl (pH85) containing 10 mM 2-mercaptoethanol and 1 mM phenylmethylsulfonylfluoride and lysed by sonication with three 20-s pulses generated by aSonifier Cell Disrupter 250 (Branson, Danbury, Conn.). After each 20-sburst, the cells are cooled for 5 min in a wet-ice bath. Cell walldebris is removed by centrifugation at 10,000×g for 10 min, and thesupernatant is applied to a 1- to 5-ml column of Ni-nitrilotriaceticacid resin (Qiagen, Chatsworth, Calif.) equilibrated by being washedwith 5 volumes of buffer A (20 mM Tris-HCl pH 8.51, 100 mM KCl, 20 mMimidazole, 10 mM 2-mercaptoethanot, 10% [vol/vol] glycerol). The columnwas washed with 10 volumes of buffer A, 2 volumes of buffer B (20 mMTris-HCl pH 8.51, 1 M KCl, 10 mM 2-mercaptoethanoL 10% [vol/vol]glycerol), and 2 volumes of buffer A to remove unbound protein.Recombinant protein containing the His tag is eluted from the column bywashing with 5 volumes of buffer C (20 mM Tris-HCl [pH 8.5], 100 mM KCl.100 mM imidazole, 10 mM 2-mercaptoethanol, 10% [vol/vol] glycerol).Recombinant proteins without the His tag are purified after directcleavage of the His tag from the column-bound protein by treatment withapproximately 1,000 U of recombinant tobacco etch virus (rTEV) proteaseper 3 mg of bound fusion protein. When this procedure is used, the rTEVprotease is added to 5 ml of buffer A supplemented with 1 mMdithiothreitol and 0.5 mM EDTA and digestion is conducted for 2 h at 30°C. on a shaking platform. The recovered proteins are concentrated to 0.5ml with Centrisep 3 or Centrisep 10 concentrators (Amicon, Beverty,Mass.), reconstituted to 3.0 ml with phosphate-buffered saline (PBS),and desalted either with desalting columns (Econopac 10 DG columns;Bio-Rad) or by dialysis against PBS with Slide-A-Lyser cassettes(Pierce, Rockford, Ill.). The concentration of the purified protein isestimated by a protein assay (Pierce), and the material is stored at 4°C. The purity of recombinant proteins is estimated by silver stainingafter sodium dodecyl sulfate-potyacrylamide gel electrophoresis(SDS-PAGE).

EXAMPLE 65 Identification and Characterization of Yersinia Superantigen

[1996] Bacterial strains, plasmids, and bacteriophage are given inMiyoshi-Akiyama T et al., J. Immunol. 154: 5228-5234 (1995). Y.pseudotuberculosis CYP86-15, serotype 4b, isolated from a patient in themass outbreak of Y. pseudotuberculosis infection in Chiba prefecture in1986, is used to prepare YPM and construct a genomic library. XEMBL3phage vector DNA and the host strain, Escherichia coli XL1-Blue MRA(P2), are purchased from Stratagene (La Jolla, Calif.). E. coli DH10B ispurchased from Life Technologies (Gaithersburg, Md.) and used as thehost strain for pUC19 and pMW119 plasmid vectors. The λ-in vitropackaging mixture is purchased from Amersham International (Bucks, U.K.)Nippon Gene Co. (Tokyo, Japan). The low copy plasmid pMW119 is purchasedfrom Nippon Gene Co. (Tokyo, Japan).

[1997] Purification of YPM is given in Miyoshi-Akiyama Tet al., supra.Briefly, Y. pseudotuberculosis strain 86-15, serotype 4b, isolated froma patient in a mass outbreak of Y. pseudotuberculosis infection inShisui Town, Chiba Prefecture, Japan, in March 1986 (4), is provided byDr. Kenji Koiwai, Chiba Institute of Health Science, Chiba, Japan, andused for YPM purification. The bacteria are grown in 20 liters of brainheart infusion broth for 18 h at 27° C. (yields 20 g wetweight/culture). Twenty grams of the bacteria are suspended in 200 ml ofPBS (pH 7.2) and ultrasonicated. The supernatant of the suspension ofdisrupted cells is depleted of nucleic acids by streptomycin treatmentand of unprecipitated substances by precipitation with 100% saturatedammonium sulfate. The precipitated mitogenically active fraction in theammonium sulfate saturation is serially chromatographed onDEAE-Sepharose, Sephacryl S-100 HR, and TSK-gel G2000SW HPLC system(Tosoh Corp., Tokyo, Japan). In SDS-PAGE analysis, the sample obtainedafter chromatography on TSK-gel G2000SW HPLC system migrate as a singleband at a m.w. of 21 kDa. The total amount of YPM, recovered is about100 to 200 μg from 20 g of wet bacteria.

[1998] Cloning and sequencing of the DNA fragment encoding the 30 aminoacid residues of the N-terminal region of YPM are given inMiyoshi-Akiyama T et al., supra. The 30 amino acid residues of theN-terminal region of YPM (T[I or D]YDNTLNSIPSLRIPN1ATYTGTIOGKGE) aredetermined by commercial services. In the second residue of theN-terminal amino acid sequence, aspartic acid [D] is identified in oneanalysis and isoleucine [I] in two analyses. Based on the sequenceincluding isoleucine in the second residue, two degenerateoligonucleotides (Primers used are given in Table I of Miyoshi-Akiyama,T. et al., J. Immunol. 154: 5228-5234 (1995)) deduced from both sides ofthe 30 residues of the N terminus of YPM are synthesized to amplify theDNA fragment encoding the YPM N terminus using the PCR method.

[1999] Genomic DNA from Y. pseudotuberculosis CYP 86-15 is isolatedaccording to the procedures described previously. The DNA was denaturedfor 5 min in a boiling bath and used as the template DNA for PCR. ThePCR was run in 50-μl reaction mixtures containing 300 ng/ml template,0.2 ng/ml RNase, 0.5 mM deoxynucleoside triphosphate mixture, 4 nM PCRprimer 1.1 nM PCR primer 2, and 40 U/ml Vent DNA polymerase (New EnglandBiolabs, Beverly, Mass.) using a DNA Thermal cyclar 480 (Perkin-Elmer,Norwalk, Conn.). The reaction mixtures are overlaid with 50 μl ofmineral oil. The PCR are subjected to the following reaction conditions:40 cycles of a combination of 30 s at 95° C., 30 s at 25° C., and 1 minat 55° C./cycle. The PCR products obtained are purified bypheniol-chloroform extraction and applied to PAGE (10% gel). The 90-basepair (bp) fragment encoding the 30 residues of the N terminus of YPM isexcised from the gel and phosphorylated by T4 polynucleotide kinase,then ligated into the SmaI site of pUC19 to obtain plasmid pUC-YPM-N90.

[2000] DNA sequencing of the gene encoding the 30 residues of the Nterminus of YPM is performed by the method of Sangar et al., Proc. Natl.Acad. Sci. 12: 5436-5442 (1977) using an ABI 377A DNA sequencer system(Applied Biosystems Japan, Tokyo, Japan) with AmpliTaq (AmershamInternational) and PRISM ready reaction dye primer cycle sequencing kitM13 Rev. (Applied Biosystems Japan). On the basis of the DNA sequence,digoxigenine-labeled probe (DIG-labeled oligonucleotide probe in TableI) is synthesized and used as the probe for screening the genomiclibrary of Y. pseudotuberculosis.

[2001] Construction and screening of a genomic library of Y.pseudotuberculosis is given in Miyoshi-Akiyama T et al., supra. Totalgenomic DNA of Y. pseudotuberculosis is partially digested with BamHI,and the resulting DNA fragments are ligated into λEMBL3/BamHI arms andpackaged into the λ phage particle. The mixture of the phage derivativesand E. coli XL1-Blue MRA(P2) in L soft agar containing 0.2% maltose and10 mM MgSO4 is plated on L broth agar plates, and the plates areincubated overnight at 37° C. Grown plaques aretransferred to Hybond N+filters (Amersham International) and screened with the DIG-labeledoligonucleotide probe by the plaque hybridization method using a DIGNucleic Acid Detection Kit (Boehringer Mannheim, Indianapolis, Ind.).Phage plaques showing positive hybridization are picked, mixed with E.coli XL1-Blue MRA(P2) in a ratio of 10:1, and cultured overnight. Therecombinant phage DNA is prepared from the culture supernatants usingpolyethylene glycol and used in the following subcloning experiment.

[2002] Subcloning and sequencing of the gene coding YPM is given inMiyoshi-Akiyama et al., supra.

[2003] The DNA preparations from the recombinant phages are digestedwith SalI and ligated into the same site of pMW119. The recombinantpMW119, pMW-YPM, is introduced into E. coli DH10B. The pMW-YPM DNA ispurified from the E. coli cells and used as template for DNA sequencing.DNA sequencing experiments are performed using a Shimadzu DNA sequencerDSQ-1 system (Shimadzu Co., Kyoto, Japan) with Sequenase Ver. 2.0(Amersham International pic.) and synthetic oligonucleotide primers(Sequence primers 1-7 in Table I). The sequence data obtained areanalyzed by the GENETYX-Mac program (SDC, Tokyo, Japan).YPM

[2004]E. coli DH10B carrying the pMW-YPM is cultured overnight at 37° C.Tween 80 (0.1%) is added to the culture supernatant obtained, and thisis applied to an anti-YPM polyclonal rabbit Ab-conjugated Sepharosecolumn (1.6×3 cm) equilibrated with PBS (pH 7.2) containing 0.1% Tween80. The elution is performed using 0.1 M glycine-Cl (pH 2.7). The elutedfractions are neutralized, pooled, desalted, concentrated, and examinedfor biologic properties as YPM.

[2005] Biologic Assays

[2006] Lymphoid cells and L cells.: In brief, human PBMC are obtainedfrom peripheral blood of healthy donors by Ficoll-Conray densitygradient centrifugation. T cell-enriched human PBMC are obtained by theS-2-aminoethylisothiouronium-treated SRBC rosette method as arosette-forming cell fraction. T cells are obtained by treatment withthe rosette-forming cell fraction with a combination of mAb to HLA-DR(12C3) and nontoxic rabbit serum. L cells transfected with HLA-DR4 genes(8124), L cells transfected with DQw1 genes (LAQ8251), L cellstransfected with DP(Cp63) genes (LAP4108), and control L cells (8400)are used as AC in T cell activation. L cells are treated with 50 μg/mlmitomycin C at 37° C. for 30 min and irradiated (3500 rad) before use.Assays of T cell responses.

[2007] Assays for YPM-induced T cell responses: In brief, in the assayfor IL-2 activity, whole human PBMC arestimulated with mitogens, and theculture supernatants are examined for IL-2 activity using IL-2-dependentCTLL-2 cells. For examination of the requirement of MHC class IImolecules on AC in T cell activation, T cells from human PBMC arestimulated with mitogens in the presence or absence of HLA class II+ Lcells or control L cells. The culture supernatants are assayed for IL-2activity.

[2008] PCR method for determining TCR-Vβ usage.: Determination of TCR-Vβselectivity of human T cells stimulated by the cloned YPM is performedby the reverse transcription-PCR method. In brief, human PBMC arestimulated with several preparations of YPM or anti-CD3 mAb, and the Tcell blasts collected are expanded with IL-2 for 2 days. Samples of cDNAsynthesized by incubating total mRNA obtained from T cell samples withreverse transcriptase are amplified by the PCR method using 22 5′Vβ-specific sense primers and 3′ Cβ-specific antisense primerend-labeled with ³²P. TCR Cα cDNA as an internal control wasco-amplified in parallel. The amplified products are separated byelectrophoresis on 2.5% agarose gel and examined for radioactivity byautoradiography and the GS-250 Molecular Imager System (Nippon Bio-RadLaboratories, Tokyo, Japan). Radioactivities in the Vβ band arenormalized to the Ca band as the Vβ:Cα ratio.

EXAMPLE 66 Superantigen DNA Delivered to Tumor Cells as Immunoconjugates

[2009] Cloning, expression, purification, and characterization of ScFvand ScFv-protamine proteins carried out by the method of Li et al CancerGene therapy 8: 555-565 (2001). The anti-ErbB2 ML39 ScFv gene sequenceis removed from pSyn-1 ML3920 and cloned into pAcGP67B baculo-virustransfer vector at restriction sites NcoI and NotI to allow secretion ofthe ScFv and ScFv fusion proteins into the medium. A six-His tail isalso engineered at the C-terminus of the peptide to facilitatepurification. The full-length human protamine sequence is describedpreviously. The sequence for the truncated form of human protaminecomprising amino acids 8 through 29 is as follows:RSQSRSRYYRQRQRSRRRRRRS. The sequence is placed downstream of the ML39ScFv, with a His 6×6 tail at the C-terminus. Recombinant baculovirus isgenerated by using the BaculoGold system from Pharmingen (San Diego,Calif.).

[2010] To isolate recombinant proteins from culture supernatants,supernatants from SF21 cell cultures infected with the recombinantviruses are collected by centrifugation at 2000 rpm for 20 minutes at 4°C. The proteins are precipitated by addition of equal volume ofsaturated ammonium sulfate to the harvested culture supernatant,followed by incubation of the mixture at 4° C. overnight. The proteinsare then pelleted by centrifugation at 10,000×g for 30 minutes at 4° C.The resulting pellets are resuspended in phosphate-buffered saline(PBS)/10% glycerol, followed by extensive dialysis with 5% glycerol/PBS.The dialyzed protein preparations are then applied to a Proboundnickel-chelating column (Invitrogen, San Diego, Calif.). RecombinantScFv proteins are purified under native condition using increasingconcentrations of imidazole in the elution buffer. The collected proteinfractions are then analyzed for purity using sodium dodecyl sulfatepolyacrylamide gel electro-phoresis (SDS-PAGE) and Western blot. Thepurified proteins are dialyzed against PBS plus 5% glycerol to removeimidazole.

[2011] To isolate protein from cellular extracts, cells are dissolved in6 M guanidine hydrochloride (GuaHCl) for 30 minutes on ice, followed bybrief ultrasonication. Insoluble cellular debris is then removed bycentritugation at 12,000×g for 30 minutes at 4° C. The resulting proteinpreparations are then applied to a Probound column (Invitrogen,Carlsbad, Calif.) under denaturing conditions (6 M GuaHCl). Recombinantproteins are then eluted by increasing concentrations of imidazole inthe presence of 6 M GuaHCl. The eluted proteins are refolded usingmethods previously described with slight modifications. Briefly, thesamples are first dialyzed against Buffer I, which consists of PBScontaining linear decreasing concentrations of GuaHCl (from 6 to 0 M)generated by a gradient maker (Fisher Scientific, Atlanta, Ga.) over aperiod of 48-72 hours, followed by dialysis against Buffer II, whichconsists of 5% glycerol, 0.5 M arginine, 1 mM EGTA, and 1 mM glutathionein reduced and oxidized form at 4° C. for 48 hours. Afterwards, thebuffer is changed to PBS/5% glycerol by dialysis. Finally, the proteinpreparations are concentrated and stored at 4° C. with 0.005% sodiumazide, or aliquoted and frozen at −70° C.

[2012] Flow cytometry: To determine binding of ScFv to the ErbB2molecules, we use ErbB2(+) cells (SKBR3) and ErbB2(−) cells (MCF7) in aflow cytometry assay. Briefly, the cells are first detached from thebottom of tissue culture dishes by using a mild method for protection ofthe cell surface proteins, i.e., incubation of the cells in enzyme-freecell dissociation buffer (Life Technologies, Rockville, Md.) for 20minutes, followed by washing with PBS containing 5% PCS. The cellsuspension is then incubated with ScFv (2 μg/mL for 10⁶ cells) for 30minutes on ice. The cells are then washed 3× with PBS/5% PCS, followedby further incubation with murine anti-His tag monoclonal antibody(Babco, Berkeley, Calif.). Finally, the cells are washed 3× with PBS/5%PCS prior to a 30-minute incubation with FITC-conjugated goat antimouseIgG. The reaction is terminated by three additional washes with PBS/5%PCS and fixed in 2% formaldehyde/PBS. Positive cells are scored by FACSanalysis.

[2013] SEA DNA Binding to Protamine-Fab fusion proteins: The SEA DNAbinding to the antiERB2-protamine fusion protein is examined by agel-shift assay. When increasing amounts of the fusion protein are mixedwith the radiolabeled DNA fragments or whole plasmid DNA decreasingamounts of DNA fragments or whole plasmid DNA migrated into the agarosegels and the DNA entering the agarose gels migrated more slowly. Incontrast, the DNA incubated with the antiERB2 protein alone show nosignificant change of its mobility in the agarose gels. The bindingactivity of the fusion protein to tumor cell surface after it wascoupled with DNA is further examined by fluorescence activated cellsorting (FACS) in which the anti-ERB2-protamine-DNA complexes showedpositive staining. Thus, the protamine fusion protein maintains bindingactivity to tumor cells when coupled with SEA DNA molecules.

[2014] Construction and expression of superantigen: The encoding gene ofSEA is chosen for the mammalian toxin expression vectors due to theaccumulated knowledge of the encoding gene sequence-functionrelationships. The gene is placed under the control of CMV and T7promoters. These constructs allow expression of the toxin by transfectedtumor cells. When the SEA expressors are transfected into mammaliantumor cells using lipofectin,22 SEA is produced and detected in themedia.

[2015] Selective secretion of SEA by Tumor Cells Targeted by fusionprotein-Superantigen DNA complexes: To investigate whetheranti-ERB2-protamine fusion protein can transfer the toxin expresser intotumor cells, the purified anti-ERB2 Fv-protamine protein is incubatedwith pCMV-SEA plasmid DNA at a ratio of 2:1 (determined by titration) in0.2 M NaCl solution to form fusion protein-DNA complexes. The targettumor cells are incubated with the anti-ERB2-Fv-protamine-SEA expressorcomplexes, SEA expresser alone or Fv-protamine fusion protein alone.After 48 h of incubation, SEA activity in the culture cells areexamined.After incubation with the anti-ERB2-Fv-protamine-toxin expressercomplexes for 48 h, the target tumor cells show a significant secretionof superantigen SEA.

[2016] DNAse protection assay: Protection of SEA DNA by protein and/orlipid is performed by treating the complex with excessive amount ofDNase (40-80 U of DNase I per microgram of DNA). After incubation at 37°C. for 2 hours, the reaction mixture is then added with SDS to finalconcentration of 1% and Proteinase K at 1 mg/mL, followed by incubationat 37° C. for 2 hours. The mixture is then twice extracted withphenol/chloroform for isolation of DNA fragments. The aqueous phasecontaining the DNA fragments following extraction is then analyzed byethidium bromide staining of 1% agarose gel and photographed under UVlight

[2017] Preparation of Superantigen-DNA-oligolysine-RGD-Complexes:

[2018] This is carried out by the method of Harbottle R P et al., HumanGene Therapy 9: 1037-1047 (1998). American firefly (Photinus pyralis)luciferase under the control of a simian virus 40 (SV40) promoter andenhancer in the vector pGL3 (Promega, Madison, Wis.) are used as areporter gene. Plasmid SEA or SEB DNA is purified using Qiagen(Chatsworth, Calif.) columns and the endotoxin extraction kit afteralkaline lysis of cells pelleted from overnight cultures of Escherichiacoli DH5or. DNA-[K]₁₆RGD complexes are prepared in microcentrifuge tubesby the addition of 0.2 μg of plasmid DNA to serial dilutions of peptidein 20 μl of OptiMem (GffiCO-BRL) or HBS. The mixtures are vortexed andincubated at room temperature for 30 min before the complexes areanalyzed on 0.8% agarose gels.

[2019] Oligolysine peptide synthesis and chemical characterization:Peptide synthesis is performed on an Applied Biosystems. Inc. (ABI.Foster City. Calif.) solid-phase batch peptide synthesizer (model 431 A)using a 200-400 mesh p-benzyloxybenzyl alcohol (HMP) resin (Novabiochem.San Diego, Calif.). High-performance liquid chromatography (HPLC) isperformed using a Gilson HPLC system composed of two solvent deliverypumps (model 306 v 1.0). a manual-Rheodyne injection valve (77251). anda variable-wavelength detector (model 118). Mass spectrometric analysisof the peptide is performed using a LASERMAT 2000 matrix-assisted laserdesorption/ionization-time of flight (MALDITOF) mass spectrometer(Thermobioanalysis. San Jose. Calif.). Amino acid composition analysesare performed using an ABI amino acid analyzer (model 420A) with on-linePTC-amino acid detection. Sequence analyses are carried out using an ABIprotein/peptide sequencer (model 477A) with on-line PTH analyzer (model120A) and data analysis system (model 610A).

[2020] The peptide N—[K]₁₆GGCRGDMFGCA ([K]₁₆RGD) is synthesized on asmall scale (0.1 mmol), using the standard fluorenylmethoxycarbonyl(FMOC)-amino acid-coupling protocol but with sequential benzoicanhydride capping steps during the generation of the N-terminalhexadeca-L-lysine sequence. Once the synthesis is concluded, the peptideis cleaved from the resin using a trifluoroacetic acid (TFA) solution (7ml) containing the following scavengers: phenol (7% w/v), thioanisole(4%, v/v), and ethanedithiol (2%, v/v). The peptide is precipitatedusing ice-cold methyl/-butyl ether (MTBE) and pelleted bycentrifugation. The pellet is then washed repeatedly with MTBE and driedin vacua. This dried pellet is dissolved in a minimum volume of aqueousTFA (0.1%, v/v), and then applied to a P2 BioGel gel-filtration column(2×28 cm) (Bio-Rad Laboratories, Hercules, Calif.). The column is elutedwith aqueous TFA (0.1%, v/v) at a flow rate of 0.5 ml/min and the eluantmonitored at 223 nm. Salt-free peptide-containing fractions are thencombined and freeze dried, giving the crude peptide (10 mg). This crudepeptide is then dissolved in aqueous acetic acid (7%, v/v; 160 ml) andthe solution buffered to pH 6.0 with aqueous ammonia solution.

[2021] Dimethyl sulfoxide is added (20% [v/v] final concentration) andthe solution stirred at ambient temperature for 24 hr. Followingfivefold dilution in water, the crude cyclized peptide is applied to areversed-phase C₁₈ Nova-Pak HPLC column (2.5×10 cm, ⁶-μη particle size)(Waters-Millipore, Bedford, Mass.) previously equilibrated with aqueousTFA (0.1%, v/v) (buffer A), and then eluted with a linear gradient (125ml) of 0-80% buffer B, which is composed of TFA (0.1%, v/v) inacetonitrile. Fractions are collected at a flow rate of 5 ml/min andelution is monitored at 223 nm. Fractions containing peptide eluting at41% buffer B are combined and freeze dried to give a lyophilized powder(7.1 mg). By matrix-assisted laser desorption/ionization-time of flight(MALDITOF) mass spectrometry, using an acetonitrile-methanol matrixcontaining 33 mM a-cyano-4-hydroxycinnamic acid (α-CHC), an [M+H]+molecular ion is observed at 3123.3 m/z. The mass calculated from theempirical formula, C₁₄H₆₃N₁₄O₁₄S₃, is expected to be 1072.2 m/z. Theamino acid composition of purified cyclized peptide is checked by aminoacid analysis. A sample of the peptide was then dissolved in 50 mMTris-HCl, pH 9.0, containing acetonitrile (10%, v/v) and digested withendoproteinase LysC (Wako Bioproducts, Wako, Tex.) for 16 hr at ambienttemperature. The digest is then subjected to amino acid sequencing andanalysis by MALDITOF mass spectrometry. An ion is observed at 1072.2m/z. The mass calculated from the empirical formula of the RGD domain,is expected to be 1072.2 m/z.

[2022] Transfection of Tumor Cells with Adenoviral Vectors encodingSuperantigen and: ttenuated Factor VII: This is carried out by themethod of Zhiwei H et al., Proc. Natl. Acad. Sci. 97: 9221-9225 (2000);Zhiwei H et al., Proc. Natl. Acad. Sci. 96: 8161-8166 (1999).

[2023] Cell Lines: LXCSN are TF are human melanoma lines. B16F10 is amouse melanoma line. The culture medium is DMEM+10% FBS for all of thetumor lines.

[2024] Preparation of the Immunoconjugates: The procedures involvestransfecting the expression vector pcDNAS.1 (Invitrogen) into CHO cellsor the expression vector pMK33/pMtHy (gift from M. Koelle, YaleUniversity) into Drosphila cells; each vector carried a cDNA encoding asecreted immunoconjugate. The cDNAs for the SEA or SEB superantigen areprepared by the method of Dow S W et al., J. Clin. Invest. 101:2406-2414(1998) is used. The genes for SEA and SEB are obtained from Dr. JohnKappler (National Jewish Hospital), and the gene for TSST-1 is obtainedfrom Dr. Brian Kotzin (National Jewish Hospital). After PCRamplification, all three genes are subcloned into a eukaryoticexpression vector (PCR3; Invitrogen Corp., San Diego, Calif.). Theprimers used for cloning

0 SEQUENCE LISTING The patent application contains a lengthy “SequenceListing” section. A copy of the “Sequence Listing” is available inelectronic form from the USPTO web site(http://seqdata.uspto.gov/sequence.html?DocID=20040214783). Anelectronic copy of the “Sequence Listing” will also be available fromthe USPTO upon request and payment of the fee set forth in 37 CFR1.19(b)(3).

What is claimed is:
 1. A method of treating a subject having a tumor or malignant mass comprising administering intratumorally to said subject a tumoricidally effective amount of a superantigen composition comprising a superantigen or a superantigen-like exotoxin.
 2. The method of claim 1 wherein the superantigen or exotoxin is selected from the group consisting of a Staphylococcal enterotoxin, a Streptococcal pyrogenic exotoxin, Yersinia pseudotuberculosis superantigen, Mycoplasma arthritides superantigen, and Clostridium perfringens superantigen-like exotoxin.
 3. A method of treating a subject having a tumor comprising administering intratumorally to said subject a tumoricidally effective amount of a superantigen composition which comprises: (a) a biologically active fragment of a full-length Staphylococcal enterotoxin, a Streptococcal pyrogenic exotoxin, Yersinia pseudotuberculosis superantigen, Mycoplasma arthritides superantigen, and Clostridium perfringens superantigen-like exotoxin; (b) a biologically active homologue of a Staphylococcal enterotoxin, a Streptococcal pyrogenic exotoxin, Yersinia pseudotuberculosis superantigen, Mycoplasma arthritides superantigen, and Clostridium perfringens superantigen-like exotoxin, (c) a biologically active fusion polypeptide comprising a superantigen sequence of (1) said full length superantigen or exotoxin, (2) said fragment or (3) said homologue which is fused to a fusion partner polypeptide sequence; wherein: (i) the fragment, homologue or fusion polypeptide has the biological activity of (A) stimulating T cells or (B) stimulating T cells via a T cell receptor Vβ region; and (ii) the homologue has the following sequence homology or identity compared to the native superantigen or exotoxin: (A) at least 20% amino acid sequence identity as measured using a sequence comparison algorithm; or (B) sequence homology characterized as a z value exceeding 10 using an algorithm and Monte Carlo analysis according to W. R. Pearson and D. J. Lipman in the Proceedings of the National Academy of Science U.S.A., 85:2444-2448, 1988, when the sequence of the homologue is compared to the sequence of a native Staphylococcal enterotoxin, a Streptococcal pyrogenic exotoxin, Yersinia pseudotuberculosis superantigen, Mycoplasma arthritides superantigen, and Clostridium perfringens superantigen-like exotoxin; or (C) has sequence homology characterized as a z value exceeding 10 using the FASTA/FASTP programs and Monte Carlo of Pearson and Lipman when compared to a native Staphylococcal enterotoxin, a Streptococcal pyrogenic exotoxin, Yersinia pseudotuberculosis superantigen, Mycoplasma arthritides superantigen, and Clostridium perfringens superantigen-like exotoxin
 4. The method of claim of any of claims 1-3 wherein the superantigen composition is administered in a sustained release vehicle.
 5. The method of claim 4 wherein the sustained release vehicle is selected from a group consisting of poly (D-, L- or DL-lactic acid/polyglycolide) polymer, ethylene-vinyl acetate, a bioerodible polyanhydride, a polyimino carbonate, sodium alginate microspheres, a hydrogel, a biodegradable amphipathic copolymer, and a collagen-poly (hema) hydrogel matrix.
 6. The method of any of claims 1-3 further comprising administering one or more chemotherapeutic drugs or prodrugs together with or after administration of said superantigen composition, wherein the chemotherapeutic drug or prodrug is or are given in a dose which is lower than a therapeutically effective dose of said drug or prodrug, which therapeutically effective dose is based on administration of said drug or prodrug in monotherapy or in combination therapy but without said superantigen composition.
 7. The method of claim 4 further comprising administering one or more chemotherapeutic drugs or prodrugs together with or after administration of said superantigen composition, wherein the chemotherapeutic drug or prodrug is or are given in a dose which is lower than a therapeutically effective dose of said drug or prodrug, which therapeutically effective dose is based on administration of said drug or prodrug in monotherapy or in combination therapy but without said superantigen composition.
 8. The method claim 6 wherein the chemotherapeutic drug is administered in a dose that is 10-95% below said therapeutically effective dose.
 9. The method claim 7 wherein the chemotherapeutic drug is administered in a dose that is 10-95% below said therapeutically effective dose.
 10. The method of claim 6 wherein said drug bis given within the 36 hours after the intratumoral administration of the superantigen composition.
 11. The method of claim 7 wherein said drug bis given within the 36 hours after the intratumoral administration of the superantigen composition.
 12. The method of claim 8 wherein said drug is first administered after the superantigen composition has been administered for two to four doses.
 13. The method of claim 9 wherein said drug is first administered after the superantigen composition has been administered for two to four doses.
 14. The method claim 6 wherein the chemotherapeutic drug is selected from the group consisting of an antimetabolite, a pyrimidine analogue, a purine analogue, an anthracycline, a vinca alkaloid, an anti-tubulin drug, an antibiotic, an alkylating agent, an epipodophyllotoxin, and a platinum-based agent.
 15. The method claim 7 wherein the chemotherapeutic drug is selected from the group consisting of an antimetabolite, a pyrimidine analogue, a purine analogue, an anthracycline, a vinca alkaloid, an anti-tubulin drug, an antibiotic, an alkylating agent, an epipodophyllotoxin, and a platinum-based agent.
 16. The method of any of claims 1-3 further comprising administering to the subject after the intratumoral administration of the superantigen composition, one or more additional cancer therapeutic agents selected from the group consisting of (a) a radiotherapeutic agent, (b) an antiangiogenic agent, (c) a cytotoxin, (d) an apoptosis-inducing agent, or (e) tumor-targeted form of any of (a)-(d). 